Recombinant monoclonal antibodies and corresponding antigens for colon and pancreatic cancers

ABSTRACT

The present invention provides for recombinant monoclonal antibodies that bind to human colorectal and pancreatic carcinoma-associated antigens, along with nucleic acid sequences encoding the antibody chains, and the amino acid sequences corresponding to the nucleic acids, and uses for these antibodies, nucleic acids and amino acids.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority of U.S. ProvisionalApplication Ser. No. 60/996,255, filed Nov. 8, 2007, which isincorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to the field of recombinant monoclonalantibodies and peptides and their uses in clinical and scientificprocedures, including diagnostic procedures, especially where suchprocesses involve the detection of human colorectal and pancreaticcarcinoma-associated antigens (CPAA), and the characterization of theepitopes recognized by said recombinant monoclonal antibodies andpeptides. The present invention also provides anti-CPAA antibodies andpeptides in the form of diagnostic compounds and/or pharmaceuticalcompositions, useful for the diagnostic and/or therapeutic methods ofthe present invention for diagnosing and/or treating colorectal andpancreatic carcinoma-associated pathologies

BACKGROUND OF THE INVENTION

According to the most recent data from the World Health Organization,from a total of fifty-eight million deaths worldwide, cancer accountedfor thirteen percent of all deaths. Deaths from cancer in the world areprojected to rise, with an estimated nine million people dying fromcancer in the year 2015 and over eleven million dying in the year 2030.Of all cancers, colorectal cancer is the third leading cause ofcancer-related deaths in the U.S., while pancreatic cancer is theeleventh most common cancer and the fourth leading cause of cancer deathin both men and women. This grim scenario shows the great need for newcancer diagnostics and therapies.

Modern technology, such as that involving the use of hybridomas, hasmade available to researchers and clinicians sources of highly specificand potent monoclonal antibodies useful in general diagnostic andclinical procedures. For example, there are now therapeutic antibodiesapproved by the FDA for the treatment of colorectal cancer, such asAVASTIN® (bevacizumab, Genentech, Inc.), ERBITUX® (cetuximab injection,ImClone Sys. Inc./Merck/Bristol-Myers Squibb), and VECTIBIX®(panitumumab, Amgen Inc.).

Yet the most important challenge in fighting cancer remains the pursuitof early diagnosis. The more advanced a cancer is when diagnosed, theless likely it is that therapy will be effective. The American CancerSociety estimates that ninety percent of Americans diagnosed with stage1 colon cancer are still alive five years after diagnosis, but onlysixty-eight percent of those diagnosed with stage 3 cancer are stillalive five years after diagnosis.

Hence, despite the advances in cancer research, there remains a need,for recombinant monoclonal antibodies useful for the early diagnosis andtreatment of colon and pancreatic carcinomas.

SUMMARY OF THE INVENTION

An object of the present invention provides for recombinant monoclonalantibodies, or portions of recombinant monoclonal antibodies (peptides)having specificity directed to antigens and epitopes of human colorectaland pancreatic carcinoma-associated antigens (CPAA). It is therefore anobject of the present invention to provide for a recombinant monoclonalantibody or a portion thereof, such as a paratope, having specificityfor CPAA proteins and peptides, such as an epitope on those proteins orpeptides.

A further object of the present invention provides for oligonucleotides,such as cDNAs, whose nucleotide sequences (genes) encode part or all ofthe heavy and light chains of the aforementioned recombinant antibodies.Accordingly, an aspect of the present invention provides for a geneencoding the variable region of a monoclonal antibody, specificallyrecognizing a CPAA, especially antigenic determinants or epitopes thatcommonly exist in a CPAA.

A further object of the present invention provides for a recombinantvector comprising the above genes. A further object of the presentinvention provides for a transformant obtained using the aboverecombinant vector.

It is a still further object of the present invention to providerecombinant antibodies specific for CPAA, wherein said antibodies aretagged with markers, making them easily isolatable as well as affordingversatility in using said antibodies for research, diagnostic, andclinical purposes. A further aspect of the invention provides for achimeric antibody that includes the variable regions of the heavy andlight chains of CPAA-specific murine antibody linked to the humanimmunoglobulin gamma-1 and kappa constant regions, respectively. Anotherobject of the present invention provides for a fully humanizedrecombinant antibody specific for CPAA. In an aspect of this embodiment,the fully humanized recombinant antibody is optimized to reduce itsimmunogenicity in humans, while maintaining its functionality.

It is another object of the present invention to provide a method ofusing the recombinant antibodies disclosed herein for research,diagnostic, and clinical uses. Particularly, an object of the presentinvention provides a diagnostic tool for the early detection of cancers,perhaps in patients without symptoms of disease. Another aspect providesfor an immunohistochemical tool for distinguishing between slow andaggressive pancreatic cancers.

Another object of the invention provides a method for promoting tumorregression or triggering the death of transformed cells comprisingadministering to a patient in need thereof an antibody, portion,fragment, peptide or derivative thereof that binds to a CPAA antigen,wherein a said antibody is administered in sufficient amounts to promotetumor regression or cell death.

Yet another object of the present invention provides for methods havingutility for in vitro, in situ and/or in vivo diagnosis and/or treatmentof animal cells, tissues or pathologies associated with the presence ofCPAA, using anti-CPAA antibodies and/or anti-CPAA peptides. The presentinvention also provides anti-CPAA antibodies and peptides in the form ofpharmaceutical and/or diagnostic compounds and/or compositions, usefulfor the diagnostic and/or therapeutic methods of the present inventionfor diagnosing and/or treating CPAA-related pathologies.

The present invention is also directed to an anti-CPAA chimeric orhumanized antibody comprising two light chains and two heavy chains,each of the chains comprising at least part of a human constant regionand at least part of a variable (V) region of non-human origin havingspecificity to a CPAA, said antibody binding with high affinity and/orhigh avidity to an inhibiting and/or neutralizing epitope ofCPAA-associated cells. The invention also includes a fragment or aderivative of such an antibody, such as one or more portions of theantibody chain, such as the heavy chain constant, joining, diversity orvariable regions, or the light chain constant, joining or variableregions. Example portions of the antibody are one or more of thecomplementarity determining regions (CDRs) of the antibody, which definethe specific binding to the CPAA.

It is a further object of the invention to characterize the CPAApeptides identified by the monoclonal antibodies or portions thereof.Such antigenic peptides may be useful in generating additionalantigen-binding ligands, or be used as vaccines or otherimmunostimulatory means.

Methods are also provided for making and using anti-CPAA antibodies andpeptides for various utilities of the present invention, such as hut notlimited to, hybridoma, recombinant or chemical synthetic methods forproducing anti-CPAA antibodies or anti-CPAA peptides according to thepresent invention; detecting CPAA in a solution or cell; inhibiting oneor more biological activities of CPAA-bearing cells in vitro, in situ orin vivo, including killing such CPAA-bearing cells. Hence, suchinhibition and killing can include treatment methods of the presentinvention for alleviating symptoms or pathologies involving CPAA-bearingcells, such as malignancies.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a tracing showing an HPLC elution profile of the Hollinshead“vaccine,” a purified preparation of colorectal and pancreatic carcinomacell membranes.

FIG. 2 presents the DNA sequence of the 16C3 murine antibody kappa lightchain. The putative ATG initiating codon is indicated inbold/underlined, and the putative TAG stop codon is indicated initalics/underlined.

FIG. 3 presents the DNA sequence of 16C3 antibody IgG heavy chain. Theputative ATG initiating codon is indicated in bold/underlined, and theputative TGA stop codon is indicated in italics/underlined.

FIG. 4 depicts the amino acid sequence of the 16C3 antibody kappa lightchain. CDR regions are presented in bold/underlined typeface.

FIG. 5 depicts the amino acid sequence of the 16C3 heavy chain. CDRregions are presented in bold/underlined typeface.

FIG. 6 presents several humanized 16C3 variable light chains. 16C3 isthe murine antibody sequence, ven16C3 has been veneered with humanframework sequences, cdr16C3 has been remodeled with human CDR aminoacids, abb16C3 represents abbreviated CDR grafting, sdr16C3 representssite determining amino acid changes, and fra16C3 represents a“Frankenstein” approach to remodeling the variable region by using acombination of various “pieces” of human variable regions. Numeralsreflect Kabat numbering.

FIG. 7 presents several humanized 163 variable heavy chains.Abbreviations are identical to those of FIG. 6.

FIG. 8 is a radiograph of a western blot analysis of various cell linesusing 16C3 antibody against the untreated 16C3 tumor antigen. TU=patientresected tumor sample (colorectal); LS=LS174; CF=CFPAC-1; AS=ASPC-1;HT=HT29.

FIG. 9 is a radiograph of a western blot representing the proteaseV8-treated 16C3 tumor antigen. Protease V8 treatment of 16C3 antigenfrom LS174 cell line and detection of the antigen using Western blot.LS=untreated antigen; V81=incubation with protease VS for 1 hour at roomtemperature (RT); V83=incubation with protease V8 for 3 hours at RT;V824=incubation with protease VS for 24 hour at RT.

FIG. 10 shows a western blot representing the PNGase-F treated 16C3tumor antigen. 16C3 antigen from CFPAC-1 was treated with PNGase-F(removes N-linked glycosylation) for various times. CFC=antigenincubated 24 hours at RT without enzyme; CF=control antigen untreated;CF1=antigen treated with enzyme 1 hour at RT; CF5=antigen treated withenzyme for 5 hours; CF24=antigen treated with enzyme for 24 hours. Thehigh molecular weight band is affected, but the low molecular weightband is not affected.

FIG. 11 shows western blot representing the 16C3 tumor antigen expressedin various fetal tissue extracts using 16C3 antibody. Lanes: 1=fetalintestine, Fx III (Hem), Aug. 22, 1972; 2=fetal intestine, Fx II (Hem),Aug. 22, 1972; 3=fetal Gut, Fx III, Feb. 26, 1973; 4=fetal Gut, Fx II,HB Nov. 1, 1972; 5=fetal Gut, Fx III, Dec. 20, 1972; 6=fetal Intestine,Fx I, Jun. 24, 1975; 7 fetal Gut, Fx I, Dec. 20, 1972; 8=fetal Gut, FxII, Mar. 1, 1973; 9=fetal Intestine Reg 2 and Reg 3A, Aug. 3, 1974.

FIG. 12 presents the amino acid sequences of an optimized, humanized16C3 antibody. Underlined, bolded amino acids indicate CDRs, “/”indicates the leader peptide/mature N-terminus junction and thevariable/constant domain junction.

DETAILED DESCRIPTION OF THE INVENTION

It should be understood that this invention is not limited to theparticular methodology, protocols, and reagents, etc., described hereinand as such may vary. The terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to limit thescope of the present invention, which is defined solely by the claims.

Unless otherwise defined, scientific and technical terms used inconnection with the antibodies described herein shall have the meaningsthat are commonly understood by those of ordinary skill in the art.Further, unless otherwise required by context, singular terms shallinclude pluralities and plural terms shall include the singular.Generally, nomenclatures utilized in connection with, and techniques of,cell and tissue culture, molecular biology, and protein and oligo- orpolynucleotide chemistry and hybridization described herein are thosewell known and commonly used in the art.

Standard techniques are used for recombinant DNA, oligonucleotidesynthesis, and tissue culture and transformation (e.g., electroporation,lipofection). Enzymatic reactions and purification techniques areperformed according to manufacturer's specifications or as commonlyaccomplished in the art or as described herein. The foregoing techniquesand procedures are generally performed according to conventional methodswell known in the art and as described in various general and morespecific references that are cited and discussed throughout the presentspecification, See e.g., Sambrook et al. MOLECULAR CLONING: LAB. MANUAL(3rd ed., Cold Spring Harbor Lab. Press, Cold Spring Harbor, N.Y.,2001). The nomenclatures utilized in connection with, and the laboratoryprocedures and techniques of, analytical chemistry, synthetic organicchemistry, and medicinal and pharmaceutical chemistry described hereinare those well known and commonly used in the art. Standard techniquesare used for chemical syntheses, chemical analyses, pharmaceuticalpreparation, formulation, and delivery, and treatment of patients.

Other than in the operating examples, or where otherwise indicated, allnumbers expressing quantities of ingredients or reaction conditions usedherein should be understood as modified in all instances by the term“about.”

All patents and other publications identified are expressly incorporatedherein by reference for the purpose of describing and disclosing, forexample, the methodologies described in such publications that might beused in connection with the present invention. These publications areprovided solely for their disclosure prior to the filing date of thepresent application. Nothing in this regard should be construed as anadmission that the inventors are not entitled to antedate suchdisclosure by virtue of prior invention or for any other reason. Allstatements as to the date or representation as to the contents of thesedocuments are based on the information available to the applicants anddoes not constitute any admission as to the correctness of the dates orcontents of these documents.

The present invention provides for recombinant monoclonal antibodies andpeptides and their uses in clinical and scientific procedures, includingdiagnostic procedures, especially where such processes involve thedetection of human colorectal and pancreatic carcinoma-associatedantigens (CPAA), and the characterization of the epitopes recognized bysaid recombinant monoclonal antibodies and peptides. The presentinvention also provides anti-CPAA antibodies and peptides in the form ofdiagnostic compounds and/or pharmaceutical compositions, useful for thediagnostic and/or therapeutic methods of the present invention fordiagnosing and/or treating colorectal and pancreaticcarcinoma-associated pathologies. One such anti-CPAA monoclonal antibodyhas been characterized previously, see U.S. Pat. No. 7,314,622. Theantigen-binding proteins described herein are novel, however.

Generally, monoclonal antibodies are used as invaluable reagents indiagnostics. In fact, due to their high specificities, they have playeda major role in deciphering the functions of various bio-molecules incryptic biosynthetic pathways. These have also become the reagents ofchoice for identification and characterization of tumor specificantigens and have become a valuable tool in the classification ofcancer.

With the advent of methods of molecular biology and recombinanttechnology, it is possible to produce antibody and antibody-likemolecules by recombinant means and thereby generate gene sequences thatcode for specific amino acid sequences found in the polypeptidestructure of the antibodies. Such antibodies can be produced by eithercloning the gene sequences encoding the polypeptide chains of saidantibodies or by direct synthesis of said polypeptide chains, withassembly of the synthesized chains to form active tetrameric (H₂ L₂)structures with affinity for specific epitopes and antigenicdeterminants. This has permitted the ready production of antibodieshaving sequences characteristic of neutralizing antibodies fromdifferent species and sources.

Regardless of the source of the antibodies, or how they arerecombinantly constructed, or how they are synthesized, in vitro or invivo, using transgenic animals, large cell cultures of laboratory orcommercial size, using transgenic plants, or by direct chemicalsynthesis employing no living organisms at any stage of the process, allantibodies have a similar overall 3 dimensional structure. Thisstructure is often given as H₂ L₂ and refers to the fact that antibodiescommonly comprise two light (L) amino acid chains and 2 heavy (H) aminoacid chains. Both chains have regions capable of interacting with astructurally complementary antigenic target. The regions interactingwith the target are referred to as “variable” or “V” regions and arecharacterized by differences in amino acid sequence from antibodies ofdifferent antigenic specificity. The variable regions of either H or Lchains contain the amino acid sequences capable of specifically bindingto antigenic targets.

As used herein, the term “antigen binding region” refers to that portionof an antibody molecule which contains the amino acid residues thatinteract with an antigen and confer on the antibody its specificity andaffinity for the antigen. The antibody region includes the “framework”amino acid residues necessary to maintain the proper conformation of theantigen-binding residues.

Within the variable regions of the H or L chains that provide for theantigen binding regions are smaller sequences dubbed “hypervariable”because of their extreme variability between antibodies of differingspecificity. Such hypervariable regions are also referred to as“complementarity determining regions” or “CDR” regions. These CDRregions account for the basic specificity of the antibody for aparticular antigenic determinant structure.

The CDRs represent non-contiguous stretches of amino acids within thevariable regions but, regardless of species, the positional locations ofthese critical amino acid sequences within the variable heavy and lightchain regions have been found to have similar locations within the aminoacid sequences of the variable chains. The variable heavy and lightchains of all antibodies each have three CDR regions, eachnon-contiguous with the others (termed L1, L2, L3, H1, H2, H3) for therespective light (L) and heavy (H) chains. The accepted CDR regions havebeen described by Kabat et al., 252 J. Biol. Chem. 6609-16 (1977), andCDR loops may be identified by applying these rules during anexamination of a linear amino acid sequence. The rules for defining theCDR-H3 loop can vary, however (see Chapter 4, ANTIBODY ENGIN. METHODS &PROTOCOLS, (Lo, ed. Humana Press, Totowa, N.J., 2004)), and the actualboundaries of some CDR-H3 loops may not be identified withoutexperimental techniques such as circular dichroism, nuclear magneticresonance, or X-ray crystallography.

In all mammalian species, antibody peptides contain constant (i.e.,highly conserved) and variable regions, and, within the latter, thereare the CDRs and the so-called “framework regions” made up of amino acidsequences within the variable region of the heavy or light chain butoutside the CDRs.

Regarding the antigenic determinate recognized by the CDR regions of theantibody, this is also referred to as the “epitope.” In other words,epitope refers to that portion of any molecule capable of beingrecognized by, and bound by, an antibody (the corresponding antibodybinding region may be referred to as a paratope). In general, epitopesconsist of chemically active surface groupings of molecules, forexample, amino acids or sugar side chains, and have specificthree-dimensional structural characteristics as well as specific chargecharacteristics.

An “antigen” is a molecule or a portion of a molecule capable of beingbound by an antibody which is additionally capable of inducing an animalto produce an antibody capable of binding to an epitope of that antigen.An antigen may have one or more than one epitope. The specific reactionreferred to above is meant to indicate that the antigen will react, in ahighly selective manner, with its corresponding antibody and not withthe multitude of other antibodies which may be evoked by other antigens.

Thus, the term “antibody” is meant to include both intact immunoglobulinmolecules as well as portions, fragments, peptides and derivativesthereof such as, for example, Fab, Fab′, F(ab′)₂, Fv, Fse, CDR regions,paratopes, or any portion or peptide sequence of the antibody that iscapable of binding an antigen or epitope. An antibody is said to be“capable of binding” a molecule if it is capable of specificallyreacting with the molecule to thereby bind the molecule to the antibody.

Antibody also includes chimeric antibodies, humanized antibodies,anti-idiotypic (anti-Id) antibodies to antibodies that can be labeled insoluble or bound form, as well as fragments, portions, regions, peptidesor derivatives thereof, provided by any known technique, such as, butnot limited to, enzymatic cleavage, peptide synthesis, or recombinanttechniques. Such antibodies of the present invention are capable ofbinding portions of CPAA or CPAA-bearing cells. Antibody fragments orportions may lack the Fe fragment of intact antibody, clear more rapidlyfrom the circulation, and may have less non-specific tissue binding thanan intact antibody. Examples of antibody fragments may be produced fromintact antibodies using methods well known in the art, for example byproteolytic cleavage with enzymes such as papain (to produce Fabfragments) or pepsin (to produce F(ab′)₂ fragments). Seer e.g., Wahl etal., 24 J. Nucl. Med. 316-25 (1983). Portions of antibodies may be madeby any of the above methods, or may be made by expressing a portion ofthe recombinant molecule. For example, the CDR region(s) of arecombinant antibody may be isolated and subcloned into the appropriateexpression vector. See, e.g., U.S. Pat. No. 6,680,053.

Clone 16C3 Oligonucleotide and Amino Acid Sequences

The present invention provides for a novel monoclonal antibody thatspecifically binds a CPAA. This monoclonal antibody, identified as“16C3”, which refers to the number assigned to its hybridoma clone.Herein, 16C3 also refers to the portion of the monoclonal antibody, theparatope or CDRs, that bind specifically with a CPAA epitope identifiedas 16C3 because of its ability to bind the 16C3 antibody. The severalrecombinant and humanized forms of 16C3 described herein may be referredto by the same name.

The present invention includes, within its scope, DNA sequences encodingthe variable regions of the light and heavy chains of the anti-CPAAantibody of the present invention. A nucleic acid sequence encoding thevariable region of the light chain of the 16C3 antibody is presented inFIG. 2. A nucleic acid sequence encoding the variable region of theheavy chain of the 16C3 antibody is presented in FIG. 3.

The present invention includes, within its scope, a peptide of the 16C3light chain comprising the amino acid sequence depicted in FIG. 4 andFIG. 12; and a peptide of the 16C3 heavy chain comprising the amino acidsequence depicted in FIG. 5 and FIG. 12. Further, the present inventionincludes the CDR regions depicted for the 16C3 kappa light chain whichare the residues underlined in FIG. 4, having the amino acids of CDR 1:GASENIYGALN (SEQ ID NO: 1); CDR 2: GASNLAD (SEQ ID NO:2); and CDR 3:QNVLSSPYT (SEQ ID NO:3); as well as the amino acids the light chainunderlined in FIG. 12, which include CDR 1: QASENIYGALN (SEQ ID NO:4);CDR 2: GASNLAT (SEQ ID NO:5); and CDR 3: QQVLSSPYT (SEQ ID NO:6). Theinvention similarly identifies the CDR regions for the heavy chain,underlined in FIG. 5, which include the amino acids for CDR 1:GYTFTDYAMH (SEQ ID NO:7); CDR 2: LISTYSGDTKYNQNFKG (SEQ ID NO:8); andCDR 3: CDYSGSRYWFAY (SEQ ID NO:9); as well as the amino acids the heavychain underlined in FIG. 12, which include CDR 1: GYTFTDYAMH (SEQ IDNO:7); CDR 2: ISTYSGDTKYNQNFQG (SEQ ID NO:10); and CDR 3: GDYSGSRYWFAY(SEQ ID NO:11).

Included also within the scope of the invention is any oligonucleotidesequence that encodes the amino acid sequence of 16C3 or a peptidethereof. Because the genetic code is degenerate, more than one codon canbe used to encode a particular amino acid. Using the genetic code, oneor more different oligonucleotides can be identified, each of whichwould be capable of encoding the amino acid. The probability that aparticular oligonucleotide will, in fact, constitute the actualXXX-encoding sequence can be estimated by considering abnormal basepairing relationships and the frequency with which a particular codon isactually used (to encode a particular amino acid) in eukaryotic orprokaryotic cells expressing an anti-CPAA antibody or portion. Such“codon usage rules” are disclosed by Lathe, et al., 183 J. Molec. Biol.1-12 (1985). Using the “codon usage rules” of Lathe, a singleoligonucleotide, or a set of oligonucleotides, that contains atheoretical “most probable” nucleotide sequence capable of encodinganti-CPAA sequences is identified.

Although occasionally an amino acid sequence can be encoded by only asingle oligonucleotide, frequently the amino acid sequence can beencoded by any of a set of similar oligonucleotides. Importantly,whereas all of the members of this set contain oligonucleotides whichare capable of encoding the peptide fragment and, thus, potentiallycontain the same oligonucleotide sequence as the gene which encodes thepeptide fragment, only one member of the set contains the nucleotidesequence that is identical to the nucleotide sequence of the gene.Because this member is present within the set, and is capable ofhybridizing to DNA even in the presence of the other members of the set,it is possible to employ the unfractionated set of oligonucleotides inthe same manner in which one would employ a single oligonucleotide toclone the gene that encodes the protein.

The oligonucleotide, or set of oligonucleotides, containing thetheoretical “most probable” sequence capable of encoding an anti-CPAAantibody or peptide including a variable or constant region is used toidentify the sequence of a complementary oligonucleotide or set ofoligo-nucleotides which is capable of hybridizing to the “most probable”sequence, or set of sequences. An oligonucleotide containing such acomplementary sequence can be employed as a probe to identify andisolate the variable or constant region anti-CPAA gene (Sambrook et al.,1989).

A suitable oligonucleotide, or set of oligonucleotides, which is capableof encoding a peptide of 16C3 (or which is complementary to such anoligonucleotide, or set of oligonucleotides) is identified (using theabove-described procedure), synthesized, and hybridized by means wellknown in the art, against a DNA or a cDNA preparation derived from cellswhich are capable of expressing anti-CPAA antibodies or variable orconstant regions thereof. Single stranded oligonucleotide moleculescomplementary to the “most probable” anti-CPAA region peptide codingsequences can be synthesized using procedures which are well known tothose of ordinary skill in the art. See Belagaje et al., 254 J. Biol.Chem. 5765-80 (1979); Maniatis et al., in MOLEC. MECH. IN CONTROL OFGENE EXPRESSION (Nierlich et al., eds., Acad. Press, NY, 1976); Wu etal., 1978; Khorana, 203 Science 614-25 (1979).

Additionally, DNA synthesis can be achieved through the use of automatedsynthesizers. Techniques of nucleic acid hybridization are disclosed bySambrook et al., 1989, and by Hayrnes et al., in NUCLEIC ACIDHYBRIDIZATION, A PRACTICAL APPROACH (IRL Press, DC 1985). Hybridizationwash conditions can include wash solution of 0.2×SSC/0.1% SDS andincubation with rotation for 10 minutes at room temperature, (lowstringency wash), wash solution of prewarmed (42° C.) 0.2×SSC/0.1% SDSand incubation with rotation for fifteen minutes at 42° C. (mediumstringency wash) and wash solution of prewarmed (68° C.) 0.1×SSC/0.1%SDS and incubation with rotation for fifteen minutes at 68° C. (highstringency wash). See Ausubel et al., ANTIBODIES: A LAB. MANUAL, (Harlow& Lane eds., Cold Spring Harbor Lab., 1988). Techniques such as, orsimilar to, those described above have successfully enabled the cloningof genes for human aldehyde dehydrogenases (Hsu et al., 82 Proc. Nat'lAcad. Sci. USA 3771-75 (1985)), fibronectin (Suzuki et al., 4 Bur. Mol.Biol. Organ. J. 2519-24 (1985)), the human estrogen receptor gene(Walter et al., 82 Proc. Nat'l Acad. Sci. USA 7889-93 (1985)),tissue-type plasminogen activator (Pennica et al., 301 Nature 214-21(1983)) and human term placental alkaline phosphatase complementary DNA(Keun et al., 82 Proc. Nat'l Acad. Sci. USA 8715-19 (1985)).

It is also intended that the antibody coding regions for use in thepresent invention could also be provided by altering existing antibodygenes using standard molecular biological techniques that result invariants (agonists) of the antibodies and peptides described herein.Such variants include, but are not limited to deletions, additions andsubstitutions in the amino acid sequence of the anti-CPAA antibodies orpeptides.

For example, one class of substitutions is conserved amino acidsubstitutions. Such substitutions are those that substitute a givenamino acid in an anti-CPAA antibody peptide by another amino acid oflike characteristics. Typically seen as conservative substitutions arethe replacements, one for another, among the aliphatic amino acids Ala,Val, Leu, and Ile; interchange of the hydroxyl residues Ser and Thr,exchange of the acidic residues Asp and Glu, substitution between theamide residues Asn and Gln, exchange of the basic residues Lys and Arg,replacements among the aromatic residues Phe, Tyr, and the like.Guidance concerning which amino acid changes are likely to bephenotypically silent is found in Bowie et al., 247 Science 1306-10(1990).

Variant or agonist anti-CPAA antibodies or peptides may be fullyfunctional or may lack function in one or more activities. Fullyfunctional variants typically contain only conservative variations orvariations in non-critical residues or in non-critical regions.Functional variants can also contain substitution of similar amino acidsthat result in no change or an insignificant change in function.Alternatively, such substitutions may positively or negatively affectfunction to some degree. Non-functional variants typically contain oneor more non-conservative amino acid substitutions, deletions,insertions, inversions, or truncation or a substitution, insertion,inversion, or deletion in a critical residue or critical region.

Amino acids that are essential for function can be identified by methodsknown in the art, such as site-directed mutagenesis or alanine-scanningmutagenesis. Cunningham et al., 244 Science 1081-85 (1989). The latterprocedure introduces single alanine mutations at every residue in themolecule. The resulting mutant molecules are then tested for biologicalactivity such as epitope binding or in vitro ADCC activity. Sites thatare critical for ligand-receptor binding can also be determined bystructural analysis such as crystallography, nuclear magnetic resonance,or photoaffinity labeling. Smith et al., 224 J. Mol. Biol. 899-904(1992); de Vos et al., 255 Science 306-12 (1992).

Moreover, polypeptides often contain amino acids other than the twenty“naturally occurring” amino acids. Further, many amino acids, includingthe terminal amino acids, may be modified by natural processes, such asprocessing and other post-translational modifications, or by chemicalmodification techniques well known in the art. Known modificationsinclude, but are not limited to, acetylation, acylation,ADP-ribosylation, amidation, covalent attachment of flavin, covalentattachment of a heme moiety, covalent attachment of a nucleotide ornucleotide derivative, covalent attachment of a lipid or lipidderivative, covalent attachment of phosphotidylinositol, cross-linking,cyclization, disulfide bond formation, demethylation, formation ofcovalent crosslinks, formation of cystine, formation of pyroglutamate,formylation, gamma carboxylation, glycosylation, GPI anchor formation,hydroxylation, iodination, methylation, myristoylation, oxidation,proteolytic processing, phosphorylation, prenylation, racemization,selenoylation, sulfation, transfer-RNA mediated addition of amino acidsto proteins such as arginylation, and ubiquitination.

Such modifications are well known to those of skill in the art and havebeen described in great detail in the scientific literature. Severalparticularly common modifications, glycosylation, lipid attachment,sulfation, gamma-carboxylation of glutamic acid residues, hydroxylationand ADP-ribosylation, for instance, are described in most basic texts,such as Proteins—Structure and Molecular Properties (2nd ed., T. E.Creighton, W.H. Freeman & Co., NY, 1993). Many detailed reviews areavailable on this subject, such as by Wold, Posttranslational CovalentModification of proteins, 1-12 (Johnson, ed., Academic Press, NY, 1983);Seifter et al. 182 Meth. Enzymol. 626-46 (1990); and Rattan et al. 663Ann. NY Acad. Sci. 48-62 (1992).

Accordingly, the antibodies and peptides of the present invention alsoencompass derivatives or analogs in which a substituted amino acidresidue is not one encoded by the genetic code, in which a substituentgroup is included pegylation as mentioned previously.

Similarly, the additions and substitutions in the amino acid sequence aswell as variations, and modifications just described may be equallyapplicable to the amino acid sequence of the CPAA antigen and/or epitopeor peptides thereof, and are thus encompassed by the present invention.As mentioned above, the genes encoding the monoclonal antibody accordingto the present invention is specifically effective in the recognition ofCPAA.

Recombinant Expression of Antibodies

Traditionally, monoclonal antibodies have been produced as nativemolecules in murine hybridoma lines. In addition to that technology,reviewed below, the present invention provides for recombinant DNAexpression of monoclonal antibodies. This allows the production ofhumanized antibodies as well as spectrum of antibody derivatives andfusion proteins in a host species of choice. More recently, theproduction of antibodies in bacteria, yeast, transgenic animals andchicken eggs have emerged as promising alternatives for hybridoma-basedproduction systems. The main advantages of transgenic animals arepotential high yields from renewable sources.

A nucleic acid sequence encoding at least one anti-CPAA antibody,portion or polypeptide of the present invention may be recombined withvector DNA in accordance with conventional techniques, includingblunt-ended or staggered-ended termini for ligation, restriction enzymedigestion to provide appropriate termini, filling in of cohesive ends asappropriate, alkaline phosphatase treatment to avoid undesirablejoining, and ligation with appropriate ligases. Techniques for suchmanipulations are disclosed, e.g., by Maniatis et al., MOLECULARCLONING, LAB. MANUAL, (Cold Spring Harbor Lab. Press, NY, 1982 and1989), and Ausubel, 1987, 1993, may be used to construct nucleic acidsequences which encode a monoclonal antibody molecule or antigen bindingregion thereof.

A nucleic acid molecule, such as DNA, is said to be “capable ofexpressing” a polypeptide if it contains nucleotide sequences whichcontain transcriptional and translational regulatory information andsuch sequences are “operably linked” to nucleotide sequences whichencode the polypeptide. An operable linkage is a linkage in which theregulatory DNA sequences and the DNA sequence sought to be expressed areconnected in such a way as to permit gene expression as anti-CPAApeptides or antibody portions in recoverable amounts. The precise natureof the regulatory regions needed for gene expression may vary fromorganism to organism, as is well known in the analogous art. See, e.g.,Sambrook et al., 1989; Ausubel et al., 1987-1993.

The present invention accordingly encompasses the expression of ananti-CPAA antibody or peptide, in either prokaryotic or eukaryoticcells. Suitable hosts include bacterial or eukaryotic hosts includingbacteria, yeast, insects, fungi, bird and mammalian cells either invivo, or in situ, or host cells of mammalian, insect, bird or yeastorigin. The mammalian cell or tissue may be of human, primate, hamster,rabbit, rodent, cow, pig, sheep, horse, goat, dog or cat origin, but anyother mammalian cell may be used.

Further, by use of for example, the yeast ubiquitin hydrolase system, invivo synthesis of ubiquitin-transmembrane polypeptide fusion proteinsmay be accomplished. The fusion proteins so produced may be processed invivo or purified and processed in vitro, allowing synthesis of ananti-CPAA antibody or polypeptide of the present invention with aspecified amino terminus sequence. Moreover, problems associated withretention of initiation codon-derived methionine residues in directyeast (or bacterial) expression maybe avoided. Sabin et al., 7(7)Bio/Technol. 705-09 (1989); Miller et al., 7(7) Bio/Technol. 698-704(1989).

Any of a series of yeast gene expression systems incorporating promoterand termination elements from the actively expressed genes coding forglycolytic enzymes produced in large quantities when yeast are grown inmediums rich in glucose can be utilized to obtain anti-CPAA antibodiesor peptides of the present invention. Known glycolytic genes can alsoprovide very efficient transcriptional control signals. For example, thepromoter and terminator signals of the phosphoglycerate kinase gene canbe utilized.

Production of anti-CPAA antibodies or peptides or functional derivativesthereof in insects can be achieved. For example, by infecting the insecthost with a baculovirus engineered to express a transmembranepolypeptide by methods known to those of skill. See Ausubel et al.,1987, 1993.

In one embodiment, the introduced nucleotide sequence will beincorporated into a plasmid or viral vector capable of autonomousreplication in the recipient host. Any of a wide variety of vectors maybe employed for this purpose. See, e.g., Ausubel et al., 1987, 1993.Factors of importance in selecting a particular plasmid or viral vectorinclude: the ease with which recipient cells that contain the vector maybe recognized and selected from those recipient cells which do notcontain the vector; the number of copies of the vector which are desiredin a particular host; and whether it is desirable to be able to“shuttle” the vector between host cells of different species.

Example prokaryotic vectors known in the art include plasmids such asthose capable of replication in E. coli (such as, for example, pBR322,ColE1, pSC101, pACYC 184, πVX). Such plasmids are, for example,disclosed by Maniatis et al., 1989; Ausubel et al, 1987, 1993. Bacillusplasmids include pC194, pC221, pT127, etc. Such plasmids are disclosedby Gryczan, in THE MOLEC. BIO. OF THE BACILLI 307-329 (Academic Press,NY, 1982). Suitable Streptomyces plasmids include pIJ101 (Kendall etal., 169 J. Bacteriol. 4177-83 (1987)), and Streptomyces bacteriophagessuch as φC31 (Chater et al., in SIXTH INT'L SYMPOSIUM ON ACTINOMYCETALESBIO. 45-54 (Akademiai Kaido, Budapest, Hungary 1986). Pseudomonasplasmids are reviewed in John et al., 8 Rev. Infect. Dis. 693-704(1986); Izaki, 33 Jpn. J. Bacteriol. 729-42 (1978); and Ausubel et al.,1987, 1993.

Alternatively, gene expression elements useful for the expression ofcDNA encoding anti-CPAA antibodies or peptides include, but are notlimited to (a) viral transcription promoters and their enhancerelements, such as the SV40 early promoter (Okayama et al., 3 Mol. Cell.Biol. 280 (1983)), Rous sarcoma virus LTR (Gorman et al., 79 Proc. Natl.Acad. Sci., USA 6777 (1982)), and Moloney murine leukemia virus LTR(Grosschedl et al., 41 Cell 885 (1985)); (b) splice regions andpolyadenylation sites such as those derived from the SV40 late region(Okayarea et al., 1983), and (c) polyadenylation sites such as in SV40(Okayama et al., 1983).

Immunoglobulin cDNA genes can be expressed as described by Liu et al.,infra, and Weidle et al., 51 Gene 21 (1987), using as expressionelements the SV40 early promoter and its enhancer, the mouseimmunoglobulin H chain promoter enhancers, SV40 late region mRNAsplicing, rabbit S-globin intervening sequence, immunoglobulin andrabbit S-globin polyadenylation sites, and SV40 polyadenylationelements.

For immunoglobulin genes comprised of part cDNA, part genomic DNA(Whittle et al., 1 Protein Engin. 499 (1987)), the transcriptionalpromoter can be human cytomegalovirus, the promoter enhancers can becytomegalovirus and mouse/human immunoglobulin, and mRNA splicing andpolyadenylation regions can be the native chromosomal immunoglobulinsequences.

In one embodiment, for expression of cDNA genes in rodent cells, thetranscriptional promoter is a viral LTR sequence, the transcriptionalpromoter enhancers are either or both the mouse immunoglobulin heavychain enhancer and the viral LTR enhancer, the splice region contains anintron of greater than 31 bp, and the polyadenylation and transcriptiontermination regions are derived from the native chromosomal sequencecorresponding to the immunoglobulin chain being synthesized. In otherembodiments, cDNA sequences encoding other proteins are combined withthe above-recited expression elements to achieve expression of theproteins in mammalian cells.

Each fused gene is assembled in, or inserted into, an expression vector.Recipient cells capable of expressing the chimeric immunoglobulin chaingene product are then transfected singly with an anti-CPAA peptide orchimeric H or chimeric L chain-encoding gene, or are co-transfected witha chimeric H and a chimeric L chain gene. The transfected recipientcells are cultured under conditions that permit expression of theincorporated genes and the expressed immunoglobulin chains or intactantibodies or fragments are recovered from the culture.

In one embodiment, the fused genes encoding the anti-CPAA peptide orchimeric H and L chains, or portions thereof are assembled in separateexpression vectors that are then used to co-transfect a recipient cell.

Each vector can contain two selectable genes, a first selectable genedesigned for selection in a bacterial system and a second selectablegene designed for selection in a eukaryotic system, wherein each vectorhas a different pair of genes. This strategy results in vectors whichfirst direct the production, and permit amplification, of the fusedgenes in a bacterial system. The genes so produced and amplified in abacterial host are subsequently used to co-transfect a eukaryotic cell,and allow selection of a co-transfected cell carrying the desiredtransfected genes.

Examples of selectable genes for use in a bacterial system are the genethat confers resistance to ampicillin and the gene that confersresistance to chloramphenicol. Selectable genes for use in eukaryotictransfectants include the xanthine guanine phosphoribosyl transferasegene (designated gpt) and the phosphotransferase gene from Tn5(designated neo).

Selection of cells expressing gpt is based on the fact that the enzymeencoded by this gene utilizes xanthine as a substrate for purinenucleotide synthesis, whereas the analogous endogenous enzyme can not.In a medium containing (1) mycophenolic acid, which blocks theconversion of inosine monophosphate to xanthine monophosphate, and (2)xanthine, only cells expressing the gpt gene can survive. The product ofneo blocks the inhibition of protein synthesis by the antibiotic G418and other antibiotics of the neomycin class.

These two selection procedures can be used simultaneously orsequentially to select for the expression of immunoglobulin chain genesintroduced on two different DNA vectors into a eukaryotic cell. It isnot necessary to include different selectable markers for eukaryoticcells; an H and an L chain vector, each containing the same selectablemarker can be co-transfected. After selection of the appropriatelyresistant cells, the majority of the clones will contain integratedcopies of both H and L chain vectors and/or anti-CPAA peptides.

Alternatively the fused genes encoding the chimeric H and L chains canbe assembled on the same expression vector.

For transfection of the expression vectors and production of thechimeric antibody, the recipient cell line may be a myeloma cell.Myeloma cells can synthesize, assemble and secrete immunoglobulinsencoded by transfected immunoglobulin genes and possess the mechanismfor glycosylation of the immunoglobulin. For example the recipient cellis the recombinant Ig-producing myeloma cell SP2/0 (ATCC #CRL 8287).SP2/0 cells produce only immunoglobulin encoded by the transfectedgenes. Myeloma cells can be grown in culture or in the peritoneal cavityof a mouse, where secreted immunoglobulin can be obtained from ascitesfluid. Other suitable recipient cells include lymphoid cells such as Blymphocytes of human or non-human origin, hybridoma cells of human ornon-human origin, or interspecies heterohybridoma cells.

The expression vector carrying a chimeric or humanized antibodyconstruct or anti-CPAA polypeptide of the present invention can beintroduced into an appropriate host cell by any of a variety of suitablemeans, including such biochemical means as transformation, transfection,conjugation, protoplast fusion, calcium phosphate-precipitation, andapplication with polycations such as diethylaminoethyl (DEAE) dextran,and such mechanical means as electroporation, direct microinjection, andmicroprojectile bombardment. Johnston et al., 240 Science 1538 (1988).

Another way of introducing DNA into lymphoid cells is byelectroporation. Potter et al., 81 P.N.A.S. USA 7161 (1984); Yoshikawaet al., 77 Jpn. J. Cancer Res. 1122-33 (1986). In this procedure,recipient cells are subjected to an electric pulse in the presence ofthe DNA to be incorporated. Typically, after transfection, cells areallowed to recover in complete medium for about 24 hours, and are thenseeded in 96-well culture plates in the presence of the selectivemedium. G418 selection is performed using about 0.4 mg/ml to 0.8 mg/mlG418. Mycophenolic acid selection utilizes about 6 μg/ml plus about 0.25mg/ml xanthine. The electroporation technique is expected to yieldtransfection frequencies of about 10⁻⁵ to about 10⁻⁴ for Sp^(2/0) cells.In the protoplast fusion method, lysozyme is used to strip cell wallsfrom catarrhal harboring the recombinant plasmid containing the chimericantibody gene. The resulting spheroplasts are fused with myeloma cellswith polyethylene glycol. The immunoglobulin genes of the presentinvention can also be expressed in nonlymphoid mammalian cells or inother eukaryotic cells, such as yeast, or in prokaryotic cells, inparticular bacteria.

Yeast provides substantial advantages over bacteria for the productionof immunoglobulin H and L chains. Yeasts carry out post-translationalpeptide modifications including glycosylation. A number of recombinantDNA strategies now exist which utilize strong promoter sequences andhigh copy number plasmids which can be used for production of thedesired proteins in yeast. Yeast recognizes leader sequences of clonedmammalian gene products and secretes peptides bearing leader sequences(i.e., pre-peptides). Hitzman et al., 11th Int'l Conference on Yeast,Genetics & Molec. Biol. (Montpelier, France, 1982).

Yeast gene expression systems can be routinely evaluated for the levelsof production, secretion and the stability of anti-CPAA peptides,antibody and assembled murine and chimeric or humanized antibodies,fragments and regions thereof. Any of a series of yeast gene expressionsystems incorporating promoter and termination elements from theactively expressed genes coding for glycolytic enzymes produced in largequantities when yeasts are grown in media rich in glucose can beutilized. Known glycolytic genes can also provide very efficienttranscription control signals. For example, the promoter and terminatorsignals of the phosphoglycerate kinase (PGK) gene can be utilized. Anumber of approaches can be taken for evaluating optimal expressionplasmids for the expression of cloned immunoglobulin cDNAs in yeast. SeeII DNA Cloning, 45-66, (Glover, ed., IRL Press, 1985).

Bacterial strains can also be utilized as hosts for the production ofantibody molecules or peptides described by this invention, E. coli K12strains such as E. coli W3110 (ATCC 27325), and other enterobacteriasuch as Salmonella typhimurium or Serratia marcescens, and variousPseudomonas species can be used.

Plasmid vectors containing replicon and control sequences which arederived from species compatible with a host cell are used in connectionwith these bacterial hosts. The vector carries a replication site, aswell as specific genes which are capable of providing phenotypicselection in transformed cells. A number of approaches can be taken forevaluating the expression plasmids for the production of murine andchimeric or humanized antibodies, fragments and regions or antibodychains encoded by the cloned immunoglobulin cDNAs in bacteria (seeGlover, 1985; Ausubel, 1987, 1993; Sambrook, 1989; Colligan, 1992-1996).

Host mammalian cells may be grown in vitro or in vivo. Mammalian cellsprovide post-translational modifications to immunoglobulin proteinmolecules including leader peptide removal, folding and assembly of Hand L chains, glycosylation of the antibody molecules, and secretion offunctional antibody protein.

Mammalian cells which can be useful as hosts for the production ofantibody proteins, in addition to the cells of lymphoid origin describedabove, include cells of fibroblast origin, such as Vero (ATCC CRL 81) orCHO-K1 (ATCC CRL 61) cells.

Many vector systems are available for the expression of cloned anti-CPAApeptides H and L chain genes in mammalian cells (see Glover, 1985).Different approaches can be followed to obtain complete H₂ L₂antibodies. As discussed above, it is possible to co-express H and Lchains in the same cells to achieve intracellular association andlinkage of H and L chains into complete tetrameric H₂ L₂ antibodiesand/or anti-CPAA peptides. The co-expression can occur by using eitherthe same or different plasmids in the same host. Genes for both H and Lchains and/or anti-CPAA peptides can be placed into the same plasmid,which is then transfected into cells, thereby selecting directly forcells that express both chains. Alternatively, cells can be transfectedfirst with a plasmid encoding one chain, for example the L chain,followed by transfection of the resulting cell line with an H chainplasmid containing a second selectable marker. Cell lines producinganti-CPAA peptides and/or H₂ L₂ molecules via either route could betransfected with plasmids encoding additional copies of peptides, H, L,or H plus L chains in conjunction with additional selectable markers togenerate cell lines with enhanced properties, such as higher productionof assembled H₂ L₂ antibody molecules or enhanced stability of thetransfected cell lines.

Additionally, plants have emerged recently as a convenient, safe andeconomical alternative main-stream expression systems for recombinantantibody production, which are based on large scale culture of microbesor animal cells. Antibodies may be expressed in plant cell culture, orplants grown conventionally. The expression in plants may be systemic,limited to susb-cellular plastids, or limited to seeds (endosperms).See, e.g., U.S. Patent Appl. Pub. No. 20030167531; U.S. Pat. No.6,080,560 and No. 6,512,162; and WO 0129242. Several plant-derivedantibodies have reached advanced stages of development, includingclinical trials (see, e.g., Biolex, NC).

Hybridoma Technology

The present invention provides for a hybridoma cell line that produces amonoclonal antibody that has a high degree of specificity and affinitytowards CPAA. The present invention relates also to variants and mutantsof the hybridoma cell lines characterized in detail above that whichoccur spontaneously or that can be produced artificially using knownmethods and that still have the characteristic properties of thestarting material, that is to say are still capable of producing theantibodies according to the invention or derivatives thereof andsecreting them into the surrounding medium.

The present invention also includes methods for the production of saidhybridoma cell lines and to methods for the production of saidmonoclonal antibodies. Clones and sub-clones of hybridoma cell lines areto be understood as being hybridomas that are produced from the startingclone by repeated cloning and that still have the features of thestarting clone that are essential to the invention.

More specifically, nucleic acid, protein or peptide molecules of theinvention may be utilized to develop monoclonal or polyclonal antibodiesthat bind CPAA. For preparation of the CPAA-binding antibodies of thepresent invention, any technique which provides for the production ofantibody molecules by continuous cell lines in culture may be used. Forexample, the hybridoma technique originally developed by Kohler andMilstein (256 Nature 495-497 (1975)) may be used. See also U.S. Pat. No.4,376,110; Ausubel et al., 1988; CURR. PROT. IMMUNOL. (Colligan et al.,eds., Greene Pub. Assoc. & Wiley Interscience NY, 1992-1996).

Another advantageous route for creating high affinity and/or highavidity human antibodies involves antigen priming of native humansplenocytes in vitro, transferral of the resultant in vitro antigenprimed splenocyte cells to an immunocompromised donor, e.g., a SCIDmouse, boosting the immunocompromised donor with antigen, isolatinghuman antibody secreting B-cells (IgG secreting) from the donor, andEBV-transforming the isolated human antibody secreting cells, asdescribed in U.S. Pat. No. 6,537,809.

Chimeric Humanized and Fully Humanized Antibodies

The antibodies of the present invention include chimeric antibodiescomprising part human and part mouse antibodies, in which the constantregion from human antibodies are cloned to a variable regions of lightand heavy chains from mouse. In some instances, 70% of the humansequences are retained. Humanized antibodies are chimeric antibodies inwhich perhaps 90% of the human antibody framework is retained, andcombined only with the murine the complementary determining regions.Fully humanized antibodies are also contemplated in the presentinvention.

Recombinant murine or chimeric murine-human or human-human antibodiesthat bind an epitope included in the amino acid sequences of CPAA can beprovided according to the present invention using known techniques basedon the teaching provided herein. See, e.g., Ausubel et al., 1987, 1992,and 1993; Sambrook et al., 1989. For example, an antibody may behumanized by grafting the desired CDRs onto a human framework accordingto EP0239400.

The DNA encoding an anti-CPAA antibody of the present invention can begenomic DNA or cDNA which encodes at least one of the heavy chainconstant region (H_(c)), the heavy chain variable region (H_(v)), thelight chain variable region (L_(v)) and the light chain constant regions(L_(c)). A convenient alternative to the use of chromosomal genefragments as the source of DNA encoding the murine V regionantigen-binding segment is the use of cDNA for the construction ofchimeric immunoglobulin genes. See e.g., Liu et al. 84 P.N.A.S., USA3439 (1987); 139 J. Immunol. 3521 (1987). The use of cDNA requires thatgene expression elements appropriate for the host cell be combined withthe gene in order to achieve synthesis of the desired protein. The useof cDNA sequences is advantageous over genomic sequences (which containintrons), in that cDNA sequences can be expressed in bacteria or otherhosts which lack appropriate RNA splicing systems.

For example, a cDNA encoding murine V and C region antigen-bindingsegments having anti-CPAA activity can be provided using known methodsbased on the use of the DNA sequences presented in FIG. 2-FIG. 5. Probesthat bind a portion of the DNA sequences presented in FIG. 2 or FIG. 3can be used to isolate DNA from hybridomas expressing anti-CPAAantibodies, fragments or regions, as presented herein, according to thepresent invention, by known methods.

Oligonucleotides representing the CPAA-binding antibodies light andheavy chains, presented in FIG. 2 and FIG. 3 useful for screening forthe presence of homologous genes and for the cloning of such genesencoding variable or constant regions of an anti-CPAA antibody. Suchprobes usually bind to DNA sequences (cDNA, genomic DNA, or any otherDNA) that encode the amino acid sequences underlined in FIG. 4 and FIG.5 to the light chain or heavy chain CDR regions which bind an epitope ofCPAA. Such techniques for synthesizing such oligonucleotides are wellknown. See e.g. Wu et al., 21 Prog. Nucl. Acids Res. Molec. Biol. 101-41(1978); Ausubel et al., 1987, 1993.

In an alternative way of cloning a polynucleotide encoding an anti-CPAAvariable or constant region, a library of expression vectors is preparedby cloning DNA or cDNA (from a cell capable of expressing an anti-CPAAantibody or variable or constant region) into an expression vector. Thelibrary is then screened for members capable of expressing a proteinwhich competitively inhibits the binding of an anti-CPAA antibody, suchas A2 or cA2, and which has a nucleotide sequence that is capable ofencoding peptides that have the same amino acid sequence as anti-CPAAantibodies or fragments thereof. In this embodiment, DNA, such as cDNA,is extracted and purified from a cell which is capable of expressing ananti-CPAA antibody or fragment. The purified cDNA is fragmentized (byshearing, endonuclease digestion, etc.) to produce a pool of DNA or cDNAfragments. DNA or cDNA fragments from this pool are then cloned into anexpression vector in order to produce a genomic library of expressionvectors whose members each contain a unique cloned DNA or cDNA fragmentsuch as in a lambda phage library, expression in prokaryotic cell (e.g.,bacteria) or eukaryotic cells, (e.g., mammalian, yeast, insect or,fungus). See, e.g., Ausubel, 1987, 1993; Harlow, 1988; Colligan,1992-1996; Nyyssonen et al. 11 Bio/Technology 591-95 (1993); Marks etal., 11 Bio/Technology 1145-49 (1993).

Once nucleic acid encoding such variable or constant anti-CPAA regionsis isolated, the nucleic acid can be appropriately expressed in a hostcell, along with other constant or variable heavy or light chainencoding nucleic acid, in order to provide recombinant monoclonalantibodies that bind CPAA with inhibitory activity. Such antibodies mayinclude a murine or human anti-CPAA variable region which contains aframework residue having complementarity determining residues which areresponsible for antigen binding. In one embodiment, an anti-CPAAvariable light or heavy chain encoded by a nucleic acid as describedabove binds an epitope of at least five amino acids. The amino acidsequences of such anti-CPAA variable light or heavy chains areunderlined in FIG. 4, FIG. 5, and FIG. 12.

Human genes which encode the constant (C) regions of the murine andchimeric antibodies, fragments and regions of the present invention canbe derived from a human fetal liver library, by known methods. Human Cregions genes can be derived from any human cell including those whichexpress and produce human immunoglobulins. The human C_(H) region can bederived from any of the known classes or isotypes of human H chains,including γ, μ, α, δ or ε, and subtypes thereof, such as G1, G2, G3 andG4. Since the H chain isotype is responsible for the various effectorfunctions of an antibody, the choice of C_(H) region will be guided bythe desired effector functions, such as complement fixation, or activityin antibody-dependent cellular cytotoxicity (ADCC). For example, theC_(H) region is derived from γ1 (IgG1), γ3 (IgG3), γ4 (IgG4), or μ(IgM). The human C_(L) region can be derived from either human L chainisotype, kappa or lambda.

Genes encoding human immunoglobulin C regions are obtained from humancells by standard cloning techniques (Sambrook et al., 1989; Ausubel etal., 1987, 1993). Human C region genes are readily available from knownclones containing genes representing the two classes of L chains, thefive classes of H chains and subclasses thereof. Chimeric antibodyfragments, such as F(ab′)₂ and Fab, can be prepared by designing achimeric H chain gene which is appropriately truncated. For example, achimeric gene encoding an H chain portion of an F(ab′)₂ fragment wouldinclude DNA sequences encoding the CH₁ domain and hinge region of the Hchain, followed by a translational stop codon to yield the truncatedmolecule.

Generally, the murine, human or murine and chimeric antibodies,fragments and regions of the present invention are produced by cloningDNA segments encoding the H and L chain antigen-binding regions of aCPAA-specific antibody, and joining these DNA segments to DNA segmentsencoding C_(H) and C_(L) regions, respectively, to produce murine, humanor chimeric immunoglobulin-encoding genes.

Thus, in one embodiment, a fused chimeric gene is created whichcomprises a first DNA segment that encodes at least the antigen-bindingregion of non-human origin, such as a functionally rearranged V regionwith joining (J) segment, linked to a second DNA segment encoding atleast a part of a human C region.

Therefore, cDNA encoding the antibody V and C regions, the method ofproducing the chimeric antibody according to the present inventioninvolves several steps, outlined below:

isolation of messenger RNA (mRNA) from the cell line producing ananti-CPAA antibody and from optional additional antibodies supplyingheavy and light constant regions; cloning and cDNA production therefrom;

preparation of a full length cDNA library from purified mRNA from whichthe appropriate V and/or C region gene segments of the L and H chaingenes can be: (i) identified with appropriate probes, (ii) sequenced,and (iii) made compatible with a C or V gene segment from anotherantibody for a chimeric antibody;

Construction of complete H or L chain coding sequences by linkage of thecloned specific V region gene segments to cloned C region gene, asdescribed above;

Expression and production of L and H chains in selected hosts, includingprokaryotic and eukaryotic cells to provide murine-murine, human-murine,human-human or human murine antibodies.

One common feature of all immunoglobulin H and L chain genes and theirencoded mRNAs is the J region. H and L chain J regions have differentsequences, but a high degree of sequence homology exists (greater than80%) among each group, especially near the C region. This homology isexploited in this method and consensus sequences of H and L chain Jregions can be used to design oligonucleotides for use as primers forintroducing useful restriction sites into the J region for subsequentlinkage of V region segments to human C region segments.

C region cDNA vectors prepared from human cells can be modified bysite-directed mutagenesis to place a restriction site at the analogousposition in the human sequence. For example, one can clone the completehuman kappa chain C (C_(k)) region and the complete human gamma-1 Cregion (C_(γ-1)). In this case, the alternative method based upongenomic C region clones as the source for C region vectors would notallow these genes to be expressed in bacterial systems where enzymesneeded to remove intervening sequences are absent. Cloned V regionsegments are excised and ligated to L or H chain C region vectors.Alternatively, the human C_(γ-1) region can be modified by introducing atermination codon thereby generating a gene sequence which encodes the Hchain portion of a Fab molecule. The coding sequences with linked V andC regions are then transferred into appropriate expression vehicles forexpression in appropriate hosts: prokaryotic or eukaryotic.

Two coding DNA sequences are said to be “operably linked” if the linkageresults in a continuously translatable sequence without alteration orinterruption of the triplet reading frame. A DNA coding sequence isoperably linked to a gene expression element if the linkage results inthe proper function of that gene expression element to result inexpression of the coding sequence. Expression vehicles include plasmidsor other vectors. Among these are vehicles carrying a functionallycomplete human C_(H) or C_(L) chain sequence having appropriaterestriction sites engineered so that any V_(H) or V_(L) chain sequencewith appropriate cohesive ends can be easily inserted therein. HumanC_(H) or C_(L) chain sequence-containing vehicles thus serve asintermediates for the expression of any desired complete H or L chain inany appropriate host.

A chimeric antibody, such as a mouse-human or human-human, willtypically be synthesized from genes driven by the chromosomal genepromoters native to the mouse H and L chain V regions used in theconstructs; splicing usually occurs between the splice donor site in themouse J region and the splice acceptor site preceding the human C regionand also at the splice regions that occur within the human C region;polyadenylation and transcription termination occur at nativechromosomal sites downstream of the human coding regions. See U.S. Pat.No. 6,835,823.

“Fully humanized antibodies” against CPAA are also contemplated in thepresent invention. Fully humanized antibodies are molecules containingboth the variable and constant region of the human immunoglobulin. Fullyhumanized antibodies can be potentially used for therapeutic use, whererepeated treatments are required for chronic and relapsing diseases suchas autoimmune diseases. One method for the preparation of fully humanantibodies consist of “humanization” of the mouse humoral immune system,i.e. production of mouse strains able to produce human Ig (Xenomice), bythe introduction of human immunoglobulin (Ig) loci into mice in whichthe endogenous Ig genes have been inactivated. The Ig loci areexceedingly complex in terms of both their physical structure and thegene rearrangement and expression processes required to ultimatelyproduce a broad immune response. Antibody diversity is primarilygenerated by combinatorial rearrangement between different V, D, and Jgenes present in the Ig loci. These loci also contain the interspersedregulatory elements, which control antibody expression, allelicexclusion, class switching and affinity maturation. Introduction ofunrearranged human Ig transgenes into mice has demonstrated that themouse recombination machinery is compatible with human genes.Furthermore, hybridomas secreting antigen specific hu-mAbs of variousisotypes can be obtained by Xenomice immunization with antigen. Fullyhumanized antibodies and methods for their production are known in theart. See, e.g., U.S. Pat. No. 7,276,239 and No. 6,835,823.

An aspect of the present invention provides for the production of ahumanized antibody, which is prepared according to the invention by aprocess which comprises maintaining a host transformed with a firstexpression vector which encodes the light chain of the humanizedantibody and with a second expression vector which encodes the heavychain of the humanized antibody under such conditions that each chain isexpressed and isolating the humanized antibody formed by assembly of thethus-expressed chains. The first and second expression vectors may bethe same vector. The invention further provides: a DNA sequence encodingthe light chain or the heavy chain of the humanized antibody; anexpression vector which incorporates a said DNA sequence; and a hosttransformed with a said expression vector.

Generating a humanized antibody from the sequences provided herein canbe practiced by those of ordinary skill in the art without undueexperimentation. In one approach, there are four general steps employedto humanize a monoclonal antibody, see, e.g., U.S. Pat. No. 5,585,089;No. 6,835,823; and No. 6,824,989. These are: (1) determining thenucleotide and predicted amino acid sequence of the starting antibodylight and heavy variable domains; (2) designing the humanized antibody,i.e., deciding which antibody framework region to use during thehumanizing process; (3) the actual humanizing methodologies/techniques;and (4) the transfection and expression of the humanized antibody.

Regarding the nucleotide and predicted amino acid sequences, there aretwo general methods for cloning a given antibody's heavy and light chainvariable domain cDNAs: (a) via a conventional cDNA library, or (b) viathe polymerase chain reaction (PCR). Both of these methods are widelyknown, see, e.g., U.S. Patent Appl. Pub. No. 20030166871. Given thenucleotide sequence of the cDNAs, it is a simple matter to translatethis information into the predicted amino acid sequence of the antibodyvariable domains. In the present instance, the nucleotide sequence ofthe light and heavy chains of the 16C3 antibody are shown in FIG. 2 andFIG. 3, respectively. The predicted amino acid sequence of the light andheavy chains of the 16C3 antibody are shown in FIG. 4 and FIG. 5,respectively.

Regarding the design of the humanized antibody, there are severalfactors to consider in deciding which human antibody sequence to useduring the humanization. The humanization of light and heavy chains areconsidered independently of one another, but the reasoning is basicallysimilar for each. This selection process is based on the followingrationale: A given antibody's antigen specificity and affinity isprimarily determined by the amino acid sequence of the variable regionCDRs. Variable domain framework residues have little or no directcontribution. The primary function of the framework regions is to holdthe CDRs in their proper spatial orientation to recognize antigen. Thus,the substitution of rodent CDRs such as those underlined in FIG. 4 orFIG. 5 into a human variable domain framework is most likely to resultin retention of their correct spatial orientation if the human variabledomain framework is highly homologous to the rodent variable domain fromwhich they originated. A human variable domain may be chosen, therefore,that is highly homologous to the rodent variable domain(s).

A suitable human antibody variable domain sequence can be selected asfollows:

-   -   1. Using a computer program, search all available protein (and        DNA) databases for those human antibody variable domain        sequences that are most homologous to the rodent antibody        variable domains. The output of a suitable program is a list of        sequences most homologous to the rodent antibody, the percent        homology to each sequence, and an alignment of each sequence to        the rodent sequence. This is done independently for both the        heavy and light chain variable domain sequences. The above        analyses are more easily accomplished if only human        immunoglobulin sequences are included.    -   2. List the human antibody variable domain sequences and compare        for homology. Primarily, the comparison is performed on length        of CDRs, except CDR3 of the heavy chain which is quite variable.        Human heavy chains and Kappa and Lambda light chains are divided        into subgroups; Heavy chain 3 subgroups, Kappa chain 4        subgroups, Lambda chain 6 subgroups. The CDR sizes within each        subgroup are similar but vary between subgroups. It is usually        possible to match a rodent antibody CDR to one of the human        subgroups as a first approximation of homology. Antibodies        bearing CDRs of similar length are then compared for amino acid        sequence homology, especially within the CDRs, but also in the        surrounding framework regions. The human variable domain which        is most homologous is chosen as the framework for humanization.

The actual humanizing methodologies and techniques are also within thegrasp of those of ordinary skill in the art. A DNA sequence encoding thedesired reshaped antibody can therefore be made beginning with the humanDNA whose CDRs it is wished to reshape. The rodent variable domain aminoacid sequence containing the desired CDRs is compared to that of thechosen human antibody variable domain sequence. The residues in thehuman variable domain are marked that need to be changed to thecorresponding residue in the rodent to make the human variable regionincorporate the rodent CDRs. There may also be residues that needsubstituting in, adding to or deleting from the human sequence.

Oligonucleotides are synthesized that can be used to mutagenize thehuman variable domain framework to contain the desired residues. Thoseoligonucleotides can be of any convenient size. One is normally onlylimited in length by the capabilities of the particular synthesizer onehas available. The method of oligonucleotide-directed in vitromutagenesis is well known.

Alternatively, humanization may be achieved using the recombinantpolymerase chain reaction (PCR) methodology of U.S. Pat. No. 5,858,725.Using this methodology, a CDR may be spliced between the frameworkregions of a human antibody. In general, the technique of U.S. Pat. No.5,858,725 can be performed using a template comprising two humanframework regions, AB and CD, and between them, the CDR which is to bereplaced by a donor CDR. Primers A and B are used to amplify theframework region CD. However, the primers B and C each also contain, attheir 5′ ends, an additional sequence corresponding to all or at leastpart of the donor CDR sequence. Primers B and C overlap by a lengthsufficient to permit annealing of their 5′ ends to each other underconditions which allow a PCR to be performed. Thus, the amplifiedregions AB and CD may undergo gene splicing by overlap extension toproduce the humanized product in a single reaction.

Alternatively, humanization may be achieved by chemical synthesis of theDNAs encoding the humanized immunoglobulin proteins, or fragmentsthereof, and using standard molecular biology techniques to amplify andsubclone the synthetic genes into an appropriate expression vector. Inthis case, multiple sense and antisense oligonucleotides withoverlapping sequences that emcompass the entire coding region of thehumanized antibody genes are chemically synthesized and purified. Theoligonucleotides are then mixed together such that the overlapping sensestrand oligonucleotides can anneal to their antisense strand partners,and the entire gene can be amplified to sufficient quantity using thepolymerase chain reaction. The target genes can then be cloned into anexpression plasmid using conveniently engineered restriction enzymes.

Several light and heavy chain sequences for the humanized 16C3 antibody,designed according to various techniques as described herein, arepresented in FIG. 6, FIG. 7, and FIG. 12. More specifically, fivedifferent designs for converting the murine 16C3 antibody to ahumanized, therapeutically useful antibody are presented. The designsare based upon structural information about known murine and humanantibody sequences. For example, referring to FIG. 6 and FIG. 7,“ven16C3” has been veneered with human framework sequences, “cdr16C3”has been remodeled with human CDR amino acids, “abb16C3” representsabbreviated CDR grafting, “sdr16C3” represents site determining aminoacid changes, and “fra16C3” represents a “Frankenstein” approach toremodeling the variable region by using a combination of various“pieces” of human variable regions. Human germline IgG sequences wereused for the framework sequences.

Additionally, as described in the Examples below, a recombinanthumanized antibody may be further optimized to decrease potentialimmunogenicity, while maintaining functional activity, for therapy inhumans. In this regard, functional activity means a polypeptide capableof displaying one or more known functional activities associated with a16C3 antibody of the invention. Such functional activities include,biological activity, and ability to bind to a ligand for a 16C3polypeptide. Additionally, a polypeptide having functional activitymeans the polypeptide exhibits activity similar, but not necessarilyidentical to, an activity of a 16C3 polypeptide of the presentinvention, including mature forms, as measured in a particular assay,such as, for example, a biological assay, with or without dosedependency. In the case where dose dependency does exist, it need not beidentical to that of the 16C3 polypeptides, but rather substantiallysimilar to the dose-dependence in a given activity as compared to the16C3 polypeptides of the present invention (i.e., the candidatepolypeptide will exhibit greater activity, or not more than about25-fold less, about 10-fold less, or about 3-fold less activity relativeto the 16C3 polypeptides of the present invention).

The optimized humanized 16C3 antibody, designated H16C3-Abb*, comprisesthe amino acid residues shown in FIG. 12. Note that some of the aminoacid residues within the antibody CDRs have been changed from thosepresent in the original murine CDRs. The different CDRs are consideredexamples of variants of each other, having functional equivalence,within the scope of the present application.

Following the mutagenesis reactions to reshape the antibody, themutagenized DNAs can be linked to an appropriate DNA encoding a light orheavy chain constant region, cloned into an expression vector, andtransfected into host cells, such as mammalian cells. These steps can becarried out in routine fashion. A reshaped antibody may therefore beprepared by a process comprising:

-   -   (a) preparing a first replicable expression vector including a        suitable promoter operably linked to a DNA sequence which        encodes at least a variable domain of an Ig heavy or light        chain, the variable domain comprising framework regions from a        human antibody and the CDRs required for the humanized antibody        of the invention;    -   (b) preparing a second replicable expression vector including a        suitable promoter operably linked to a DNA sequence which        encodes at least the variable domain of a complementary Ig light        or heavy chain, respectively;    -   (c) transforming a cell line with the first or both prepared        vectors; and    -   (d) culturing said transformed cell line to produce said altered        antibody.

The DNA sequence in step (a) may encode both the variable domain and/oreach constant domain of the human antibody chain. The humanized antibodycan be prepared using any suitable recombinant expression system. Thecell line that is transformed to produce the altered antibody may be aChinese Hamster Ovary (CHO) cell line or an immortalized mammalian cellline, which is advantageously of lymphoid origin, such as a myeloma,hybridoma, trioma or quadroma cell line. The cell line may also comprisea normal lymphoid cell, such as a B-cell, which has been immortalized bytransformation with a virus, such as the Epstein-Barr virus. Forexample, the immortalized cell line is a myeloma cell line or aderivative thereof.

The CHO cells used for expression of the antibodies according to theinvention may be dihydrofolate reductase (dhfr) deficient and sodependent on thymidine and hypoxanthine for growth. See Urlaub et al.,77 P.N.A.S. U.S.A. 4216-20 (1980). The parental dhfr CHO cell line istransfected with the DNA encoding the antibody and dhfr which enablesselection of CHO cell transfectants of dhfr positive phenotype.Selection is carried out by culturing the colonies on media devoid ofthymidine and hypoxanthine, the absence of which prevents untransfectedcells from growing and transformed cells from resalvaging the folatepathway and thus bypassing the selection system. These transfectantsusually express low levels of the DNA of interest by virtue ofco-integration of transfected DNA of interest and DNA encoding dhfr. Theexpression levels of the DNA encoding the antibody may be increased byamplification using methotrexate (MTX). This drug is a direct inhibitorof the enzyme dhfr and allows isolation of resistant colonies whichamplify their dhfr gene copy number sufficiently to survive under theseconditions. Since the DNA sequences encoding dhfr and the antibody areclosely linked in the original transfectants, there is usuallyconcomitant amplification, and therefore increased expression of thedesired antibody.

Another expression system for use with CHO or myeloma cells is theglutamine synthetase (GS) amplification system described in, forexample, U.S. Pat. No. 5,122,464. This system involves the transfectionof a cell with DNA encoding the enzyme GS and with DNA encoding thedesired antibody. Cells are then selected which grow in glutamine freemedium and can thus be assumed to have integrated the DNA encoding CS.These selected clones are then subjected to inhibition of the enzyme GSusing methionine sulphoximine (Msx). The cells, in order to survive,will amplify the DNA encoding GS with concomitant amplification of theDNA encoding the antibody.

Although the cell line used to produce the humanized antibody may be amammalian cell line, any other suitable cell line, such as a bacterialcell line or a yeast cell line, may alternatively be used. For example,in instances requiring no in vivo post-translational modification (suchas instances where glycosylation is not required), it is envisaged thatE. coli-derived bacterial strains could be used. The antibody obtainedis checked for functionality. If functionality is lost, it is necessaryto return to step (2) and alter the framework of the antibody.

Once expressed, the whole antibodies, their dimers, individual light andheavy chains, or other immunoglobulin forms of the present invention canbe recovered and purified by known techniques, e.g., immunoabsorption orimmunoaffinity chromatography, chromatographic methods such as HPLC(high performance liquid chromatography), ammonium sulfateprecipitation, gel electrophoresis, or any combination of these. Seegenerally, Scopes, PROT. PURIF. (Springer-Verlag, NY, 1982).Substantially pure immunoglobulins of at least about 90% to 95%homogeneity are advantageous, as are those with 98% to 99% or morehomogeneity, particularly for pharmaceutical uses. Once purified,partially or to homogeneity as desired, a humanized antibody may then beused therapeutically or in developing and performing assay procedures,immunofluorescent stainings, and the like. See generally, Vols. I & IIImmunol. Meth. (Lefkovits & Pernis, eds., Acad. Press, NY, 1979 and1981).

Phage Libraries and Alternative Recombinant Expression Systems

Along with the above production techniques, in vitro systems such asphage display methods of fully human antibodies and antibody peptides,many of the benefits of human antibodies as both diagnostics andtherapeutics are now being realized.

The recombinant antibody and its sequences of the present inventionallows for the construction of a myriad of derivatives and ligandbinding molecules with anti-PCAA binding activity. For example, the CDRsmay be recombined with an antibody library such as the n-CoDeR humanscFV library to create highly specific and functional antibodyfragments. See Moore, 426 Nature, 725-31 (2003).

A library of fully human antibodies or portions thereof may also becreated following the cloning methods based on site specific cleavage ofsingle-stranded DNAs as described by U.S. Patent Appl. Pub. No.20030232333.

Another ligand binding molecule that may be constructed from the DNAsequence information contained herein, and the associated knowledgegained about the PCAA epitopes provided by the invention herein,involves the construction of ANTICALINS® lipocalins, a widespread groupof small and robust proteins that are usually involved in thephysiological transport or storage of chemically sensitive or insolublecompounds. Several natural lipocalins occur in human tissues or bodyliquids. Despite low mutual sequence homology, the lipocalins share astructurally conserved β-barrel supporting four loops at one end, whichform the entrance to a binding pocket. The loops exhibit largeconformational differences between individual lipocalins and give riseto the variety of natural ligand specificities. This proteinarchitecture is reminiscent of immunoglobulins, with their hypervariableloops on top of a rigid framework. Unlike antibodies or some antibodyfragments, lipocalins are composed of a single polypeptide chain with160 to 180 amino acid residues, being just marginally bigger than asingle immunoglobulin domain. The set of four loops that makes up thebinding pocket shows structural plasticity and tolerates a variety ofside chains. The binding site can thus be reshaped in order to recognizeprescribed target molecules of different shape with high affinity andspecificity. ANTICALINS® lipocalins have been engineered that recognizehapten-like compounds, peptides, and protein targets, e.g. extracellulardomains of cell surface receptors. Fusion proteins with enzymes and alsobispecific binding proteins (so-called DUOCALINS® bispecific bindingproteins, Pieris AG, Freising-Weihenstephan, Germany) have also beensuccessfully prepared. Pre-clinical experiments have been conducted.See, e.g., Komdörfer et al., 330 J. Mol. Biol. 385-96 (2003).

Another antibody type with application to the invention described hereininclude the camilid immunoglobulins which possess functional heavychains and lack light chains. These antibodies are assembled fromdedicated V and C gamma genes. They have been cloned and adapted usingphage display technology to produce antigen-specific single-domainantibody fragments with intrinsic high stability. U.S. Patent Appl. Pub.No. 20030088074.

Another relevant derivative takes advantage of new technology forproviding bacterially produced antibody fragments that can crosslinkantigen and antibody effector molecules (Fc-region molecules), calledPEPBODIES™ antibody fragments. See U.S. Patent Appl. Pub. No.20040101905. Hence, the binding molecules comprising the antigen bindingsite of the anti-PCAA site is genetically fused to peptides that displayone or more of the effector functions associated with the Fc-region, andprovides for functions such as interaction with cell receptors andcomplement activation.

The new antigen receptor (IgNAR) molecules from sharks may also beconsidered a “derivative” antibody molecule. The NAR is a disulphidebonded dimer of two protein chains, each containing one variable andfive constant domains, and functions as an antibody. Nuttall et al., 270Eur. J. Biochem., 3543-54 (2003). The sequences of the PCAA-bindingantibody of the present invention may be constructed into the NARvariable region to create an in vitro library incorporating syntheticthe CDR regions. This results in a single domain binding reagent.

One of the recent advances in cancer cell biology entails the discoveryof progenitor cell lines that may exhibit cancer-cell markers. Forexample, human pancreatic epithelial progenitor cells have beenidentified and grown in culture. These cells may then be used for thegeneration of antigens useful, inter alia, for the development ofmonoclonal antibodies. U.S. Pat. No. 6,436,704. Thus, the PCAA-bindingantibody may be used to identify progenitor cells. These progenitorcells can be used as an immunogen that is administered to a heterologousrecipient, such as a mouse, for derivation of further lines ofPCAA-binding antibodies.

In conclusion, the oligonucleotide and amino acid sequences providedherein enable a myriad of possible molecules with CPAA-binding activity,and the scope of the present invention is not limited by the methods ofachieving those molecules.

Antibody Derivatives

A “derivative” of an antibody contains additional chemical moieties notnormally a part of the protein. Covalent modifications of the proteinare included within the scope of this invention. Such modifications maybe introduced into the molecule by reacting targeted amino acid residuesof the antibody with an organic derivatizing agent that is capable ofreacting with selected side chains or terminal residues. For example,derivatization with bifunctional agents, well-known in the art, isuseful for cross-linking the antibody or fragment to a water-insolublesupport matrix or to other macromolecular carriers.

Derivatives also include radioactively labeled monoclonal antibodiesthat are labeled. For example, with radioactive iodine (¹²⁵I, ¹³¹I),carbon (¹⁴C), sulfur (³⁵S), indium (¹¹¹In), tritium (³H) or the like;conjugates of monoclonal antibodies with biotin or avidin, with enzymes,such as horseradish peroxidase, alkaline phosphatase, β-D-galactosidase,glucose oxidase, glucoamylase, carboxylic acid anhydrase, acetylcholineesterase, lysozyme, malate dehydrogenase or glucose 6-phosphatedehydrogenase; and also conjugates of monoclonal antibodies withbioluminescent agents (such as luciferase), chemoluminescent agents(such as acridine esters) or fluorescent agents (such asphycobiliproteins). An example of a derivative of the antibody of theinvention is an antibody-small molecule drug conjugate, such as anantibody-maytansinoid conjugate, that displays cytotoxic activity. SeeU.S. Patent Appl. Pub. No. 20040039176. Preclinical evaluation has shownthat this conjugate acts as a tumor-activated prodrug that exhibitspotent antitumor activity in xenograft models. Further cytotoxicantibody derivatives are discussed below.

Another derivative bifunctional antibody of the present invention is abispecific antibody, generated by combining parts of two separateantibodies that recognize two different antigenic groups. This may beachieved by crosslinking or recombinant techniques. Additionally,moieties may be added to the antibody or a portion thereof to increasehalf-life in vivo (e.g., by lengthening the time to clearance from theblood stream. Such techniques include, for example, adding PEG moieties(also termed pegilation), and are well-known in the art. See U.S.Patent. Appl. Pub. No. 20030031671.

Anti-Idiotype Abs

In addition to monoclonal or chimeric anti-CPAA antibodies, the presentinvention is also directed to an anti-idiotypic (anti-Id) antibodyspecific for the anti-CPAA antibody of the invention. An anti-Idantibody is an antibody which recognizes unique determinants generallyassociated with the antigen-binding region of another antibody. Theantibody specific for CPAA is termed the idiotypic or Id antibody. Theanti-Id can be prepared by immunizing an animal of the same species andgenetic type (e.g., mouse strain) as the source of the Id antibody withthe Id antibody or the antigen-binding region thereof. The immunizedanimal will recognize and respond to the idiotypic determinants of theimmunizing antibody and produce an anti-Id antibody. The anti-Idantibody can also be used as an “immunogen” to induce an immune responsein yet another animal, producing a so-called anti-anti-Id antibody. Theanti-anti-Id can be epitopically identical to the original antibodywhich induced the anti-Id. Thus, by using antibodies to the idiotypicdeterminants of a mAb, it is possible to identify other clonesexpressing antibodies of identical specificity.

Accordingly, monoclonal antibodies generated against CPAA according tothe present invention can be used to induce anti-Id antibodies insuitable animals, such as BALB/c mice. Spleen cells from such immunizedmice can be used to produce anti-Id hybridomas secreting anti-Id mAbs.Further, the anti-Id mAbs can be coupled to a carrier such as keyholelimpet hemocyanin (KLH) and used to immunize additional BALB/c mice.Sera from these mice will contain anti-anti-Id antibodies that have thebinding properties of the original mAb specific for a CPAA epitope.

Idiotypes, Anti-Idiotypes

Additionally, antibodies against CPAA, its analogs, portions, fragments,peptides or derivatives thereof may be used to induce anti-Id antibodiesin suitable animals, such as BALB/c mice. Spleen cells from suchimmunized mice are used to produce anti-Id hybridomas secreting anti-Idmonoclonal antibodies. Further, the anti-Id antibodies can be coupled toa carrier such as keyhole limpet hemocyanin (KLH) and used to immunizeadditional BALB/c mice. Sera from these mice will contain anti-anti-Idantibodies that have the binding properties of the original monoclonalantibody specific for an epitope of CPAA, or analogs, fragments andderivatives thereof. The anti-Id antibodies thus have their ownidiotypic epitopes, or “idiotopes” structurally similar to the epitopebeing evaluated.

An anti-idiotypic (anti-Id) antibody is an antibody that recognizesunique determinants generally associated with the antigen-binding siteof an antibody. An Id antibody can be prepared by immunizing an animalof the same species and genetic type (e.g., mouse strain) as the sourceof the mAb with the mAb to which an anti-Id is being prepared. Theimmunized animal will recognize and respond to the idiotypicdeterminants of the immunizing antibody by producing an antibody tothese idiotypic determinants (the anti-Id antibody). See, e.g., U.S.Pat. No. 4,699,880 and U.S. Pat. No. 6,835,823. The anti-Id antibody mayalso be used as an “immunogen” to induce an immune response in yetanother animal, producing a so-called anti-anti-Id antibody. Theanti-anti-Id may be epitopically identical to the original mAb whichinduced the anti-Id. Thus, by using antibodies to the idiotypicdeterminants of a mAb, it is possible to identify other clonesexpressing antibodies of identical specificity.

Structural Analogs of Anti-CPAA Antibodies and Anti-CPAA Peptides

Structural analogs of anti-CPAA antibodies and peptides of the presentinvention are provided by known method steps based on the teaching andguidance presented herein.

Knowledge of the three-dimensional structures of proteins is crucial inunderstanding how they function. The three-dimensional structures ofhundreds of proteins are currently available in protein structuredatabases (in contrast to the thousands of known protein sequences insequence databases). Analysis of these structures shows that they fallinto recognizable classes of motifs. It is thus possible to model athree-dimensional structure of a protein based on the protein's homologyto a related protein of known structure. Many examples are known wheretwo proteins that have relatively low sequence homology, can have verysimilar three dimensional structures or motifs.

In recent years it has become possible to determine the threedimensional structures of proteins of up to about 15 kDa by nuclearmagnetic resonance (NMR). The technique requires a concentrated solutionof pure protein: no crystals or isomorphous derivatives are needed. Thestructures of a number of proteins have been determined by this method.The details of NMR structure determination are well-known in the art.See, e.g., Wuthrich, NMR OF PROTEINS & NUCLEIC ACIDS (Wiley, N.Y.,1986); Wuthrich, 243 Science 45-50 (1989); Clore et al., 24 CriticalRev. Biochem. Molec. Biol. 479-564 (1989); Cooke et al., 8 Bioassays52-56 (1988).

In applying this approach, a variety of ¹H NMR 2D data sets arecollected for anti-CPAA antibodies and/or anti-CPAA peptides of thepresent invention. These are of two main types. One type, COSY(Correlated Spectroscopy) identifies proton resonances that are linkedby chemical bonds. These spectra provide information on protons that arelinked by three or less covalent bonds. NOESY (nuclear Overhauserenhancement spectroscopy) identifies protons which are close in space(less than 0.5 nm). Following assignment of the complete spin system,the secondary structure is defined by NOESY. Cross peaks (nuclearOverhauser effects or NOE's) are found between residues that areadjacent in the primary sequence of the peptide and can be seen forprotons less than 0.5 nm apart. The data gathered from sequential NOE'scombined with amide proton coupling constants and NOE's fromnon-adjacent amino acids that are adjacent to the secondary structure,are used to characterize the secondary structure of the peptides. Asidefrom predicting secondary structure, NOE's indicate the distance thatprotons are in space in both the primary amino acid sequence and thesecondary structures. Tertiary structure predictions are determined,after all the data are considered, by a “best fit” extrapolation.

Types of amino acids are first identified using through-bondconnectivities. Next, specific amino acids are assigned usingthrough-space connectivities to neighboring residues, together with theknown amino acid sequence. Structural information is then tabulated andis of three main kinds: The NOE identifies pairs of protons which areclose in space, coupling constants give information on dihedral anglesand slowly exchanging amide protons give information on the position ofhydrogen bonds. The restraints are used to compute the structure using adistance geometry type of calculation followed by refinement usingrestrained molecular dynamics. The output of these computer programs isa family of structures which are compatible with the experimental data(i.e. the set of pairwise <0.5 nm distance restraints). The better thatthe structure is defined by the data, the better the family ofstructures can be superimposed, (i.e., the better the resolution of thestructure). In the better defined structures using NMR, the position ofmuch of the backbone (i.e. the amide, Cα and carbonyl atoms) and theside chains of those amino acids that lie buried in the core of themolecule can be defined as clearly as in structures obtained bycrystallography. The side chains of amino acid residues exposed on thesurface are frequently less well defined, however. This probablyreflects the fact that these surface residues are more mobile and canhave no fixed position. (In a crystal structure this might be seen asdiffuse electron density).

Thus, according to the present invention, use of NMR spectroscopic datais combined with computer modeling to arrive at structural analogs of atleast portions of anti-CPAA antibodies and peptides based on astructural understanding of the topography. Using this information, oneof ordinary skill in the art will know how to achieve structural analogsof anti-CPAA antibodies or peptides, such as by rationally-based aminoacid substitutions allowing the production of peptides in which the CPAAbinding affinity or avidity is modulated in accordance with therequirements of the expected therapeutic or diagnostic use of themolecule, for example, the achievement of greater specificity for CPAAbinding.

Alternatively, compounds having the structural and chemical featuressuitable as anti-CPAA therapeutics and diagnostics provide structuralanalogs with selective CPAA affinity, Molecular modeling studies of CPAAbinding compounds, such as CPAA receptors, anti-CPAA antibodies, orother CPAA binding molecules, using a program such as MacroModel®(Schrödinger, LLC, NY), Insight®II and Discover® (Accelrys SoftwareInc., Burlington, Mass.), provide such spatial requirements andorientation of the anti-CPAA Abs and/or peptides according to thepresent invention. Such structural analogs of the present invention thusprovide selective qualitative and quantitative anti-CPAA activity invitro, in situ and/or in vivo.

Diagnostic Applications

The present invention also provides the above anti-CPAA antibodies andpeptides for use in diagnostic methods for detecting CPAA in patientsknown to be or suspected of having pancreatic or colon carcinoma. Inanother aspect of the invention, the antibodies may detect molecularmarkers in morphologically normal cells to provide for early detectionscreening of disease-free individuals.

Anti-CPAA antibodies and/or peptides of the present invention are usefulfor immunoassays which detect or quantitate CPAA, or anti-CPAAantibodies, in a sample. An immunoassay for CPAA typically comprisesincubating a clinical or biological sample in the presence of adetectably labeled high affinity (or high avidity) anti-CPAA antibody orpolypeptide of the present invention capable of selectively binding toCPAA, and detecting the labeled peptide or antibody which is bound in asample. Various clinical assay procedures are well known in the art.See, e.g., IMMUNOASSAYS FOR THE 80'S (Voller et al., eds., Univ. Park,1981). Such samples include tissue biopsy, blood, serum, and fecalsamples, or liquids collected from the colorectal track following enema,colonoscopy, or oral laxative solution and subjected to ELISA analysisas described below.

Thus, an anti-CPAA antibody or polypeptide can be fixed tonitrocellulose, or another solid support which is capable ofimmobilizing cells, cell particles or soluble proteins. The support canthen be washed with suitable buffers followed by treatment with thedetectably labeled CPAA-specific peptide or antibody. The solid phasesupport can then be washed with the buffer a second time to removeunbound peptide or antibody. The amount of bound label on the solidsupport can then be detected by known method steps.

“Solid phase support” or “carrier” refers to any support capable ofbinding peptide, antigen, or antibody. Well-known supports or carriers,include glass, polystyrene, polypropylene, polyethylene,polyvinylidenefluoride (PVDF), dextran, nylon, amylases, natural andmodified celluloses, polyacrylamides, agaroses, and magnetite. Thenature of the carrier can be either soluble to some extent or insolublefor the purposes of the present invention. The support material can havevirtually any possible structural configuration so long as the coupledmolecule is capable of binding to CPAA or an anti-CPAA antibody. Thus,the support configuration can be spherical, as in a bead, orcylindrical, as in the inside surface of a test tube, or the externalsurface of a rod. Alternatively, the surface can be flat, such as asheet, culture dish, test strip, etc. For example, supports may includepolystyrene beads. Those skilled in the art will know many othersuitable carriers for binding antibody, peptide or antigen, or canascertain the same by routine experimentation.

Well known method steps can determine binding activity of a given lot ofanti-CPAA peptide and/or antibody. Those skilled in the art candetermine operative and optimal assay conditions by routineexperimentation.

Detectably labeling a CPAA-specific peptide and/or antibody can beaccomplished by linking to an enzyme for use in an enzyme immunoassay(EIA), or enzyme-linked immunosorbent assay (ELISA). The linked enzymereacts with the exposed substrate to generate a chemical moiety whichcan be detected, for example, by spectrophotometric, fluorometric or byvisual means. Enzymes which can be used to detectably label theCPAA-specific antibodies of the present invention include, but are notlimited to, malate dehydrogenase, staphylococcal nuclease,delta-5-steroid isomerase, yeast alcohol dehydrogenase,alpha-glycerophosphate dehydrogenase, triose phosphate isomerase,horseradish peroxidase, alkaline phosphatase, asparaginase, glucoseoxidase, beta-galactosidase, ribonuclease, urease, catalase,glucose-6-phosphate dehydrogenase, glucoamylase andacetylcholinesterase.

By radioactively labeling the CPAA-specific antibodies, it is possibleto detect CPAA through the use of a radioimmunoassay (RIA). See Work etal., LAB. TECHNIQUES & BIOCHEM. 1N MOLEC. Bio. (No. Holland Pub. Co.,NY, 1978). The radioactive isotope can be detected by such means as theuse of a gamma counter or a scintillation counter or by autoradiography.Isotopes which are particularly useful for the purpose of the presentinvention include: ³H, ¹²⁵I, ¹³¹I, ³⁵S, ¹⁴C, and ¹²⁵I.

It is also possible to label the CPAA-specific antibodies with afluorescent compound. When the fluorescent labeled antibody is exposedto light of the proper wave length, its presence can then be detecteddue to fluorescence. Among the most commonly used fluorescent labellingcompounds are fluorescein isothiocyanate, rhodamine, phycoerythrin,phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.

The CPAA-specific antibodies can also be delectably labeled usingfluorescence-emitting metals such as ¹²⁵Eu, or others of the lanthanideseries. These metals can be attached to the CPAA-specific antibody usingsuch metal chelating groups as diethylenetriaminepentaacetic acid (DTPA)or ethylenediamine-tetraacetic acid (EDTA).

The CPAA-specific antibodies also can be detectably labeled by couplingto a chemiluminescent compound. The presence of the chemiluminescentlylabeled antibody is then determined by detecting the presence ofluminescence that arises during the course of a chemical reaction.Examples of useful chemiluminescent labeling compounds are luminol,isoluminol, theromatic acridinium ester, imidazole, acridinium salt andoxalate ester.

Likewise, a bioluminescent compound can be used to label theCPAA-specific antibody, portion, fragment, polypeptide, or derivative ofthe present invention. Bioluminescence is a type of chemiluminescencefound in biological systems in which a catalytic protein increases theefficiency of the chemiluminescent reaction. The presence of abioluminescent protein is determined by detecting the presence ofluminescence. Important bioluminescent compounds for purposes oflabeling are luciferin, luciferase and aequorin.

Detection of the CPAA-specific antibody, portion, fragment, polypeptide,or derivative can be accomplished by a scintillation counter, forexample, if the detectable label is a radioactive gamma emitter, or by afluorometer, for example, if the label is a fluorescent material. In thecase of an enzyme label, the detection can be accomplished bycolorometric methods which employ a substrate for the enzyme. Detectioncan also be accomplished by visual comparison of the extent of enzymaticreaction of a substrate in comparison with similarly prepared standards.

For the purposes of the present invention, the CPAA which is detected bythe above assays can be present in a biological sample. Any samplecontaining CPAA may be used. For example, the sample is a biologicalfluid such as, for example, blood, serum, lymph, urine, feces,inflammatory exudate, cerebrospinal fluid, amniotic fluid, a tissueextract or homogenate, and the like. The invention is not limited toassays using only these samples, however, it being possible for one ofordinary skill in the art, in light of the present specification, todetermine suitable conditions which allow the use of other samples.

In situ detection can be accomplished by removing a histologicalspecimen from a patient, and providing the combination of labeledantibodies of the present invention to such a specimen. The antibody (orportion thereof) may be provided by applying or by overlaying thelabeled antibody (or portion) to a biological sample. Through the use ofsuch a procedure, it is possible to determine not only the presence ofCPAA hut also the distribution of CPAA in the examined tissue. Using thepresent invention, those of ordinary skill will readily perceive thatany of a wide variety of histological methods (such as stainingprocedures) can be modified in order to achieve such in situ detection.

The antibody, fragment or derivative of the present invention can beadapted for utilization in an immunometric assay, also known as a“two-site” or “sandwich” assay. In a typical immunometric assay, aquantity of unlabeled antibody (or fragment of antibody) is bound to asolid support that is insoluble in the fluid being tested and a quantityof detectably labeled soluble antibody is added to permit detectionand/or quantification of the ternary complex formed between solid-phaseantibody, antigen, and labeled antibody.

Typical, immunometric assays include “forward” assays in which theantibody bound to the solid phase is first contacted with the samplebeing tested to extract the CPAA from the sample by formation of abinary solid phase antibody-CPAA complex. After a suitable incubationperiod, the solid support is washed to remove the residue of the fluidsample, including unreacted CPAA, if any, and then contacted with thesolution containing a known quantity of labeled antibody (whichfunctions as a “reporter molecule”). After a second incubation period topermit the labeled antibody to complex with the CPAA bound to the solidsupport through the unlabeled antibody, the solid support is washed asecond time to remove the unreacted labeled antibody. This type offorward sandwich assay can be a simple “yes/no” assay to determinewhether CPAA is present or can be made quantitative by comparing themeasure of labeled antibody with that obtained for a standard samplecontaining known quantities of CPAA. Such “two-site” or “sandwich”assays are described by Wide, in RADIOIMMUNE ASSAY METHODS, 199-206(Kirkham, ed., Livingstone, Edinburgh, 1970).

Other types of “sandwich” assays, which can also be useful with CPAA,are the so-called “simultaneous” and “reverse” assays. A simultaneousassay involves a single incubation step wherein the antibody bound tothe solid support and labeled antibody are both added to the samplebeing tested at the same time. After the incubation is completed, thesolid support is washed to remove the residue of fluid sample anduncomplexed labeled antibody. The presence of labeled antibodyassociated with the solid support is then determined as it would be in aconventional “forward” sandwich assay.

In the “reverse” assay, stepwise addition first of a solution of labeledantibody to the fluid sample followed by the addition of unlabeledantibody bound to a solid support after a suitable incubation period, isutilized. After a second incubation, the solid phase is washed inconventional fashion to free it of the residue of the sample beingtested and the solution of unreacted labeled antibody. The determinationof labeled antibody associated with a solid support is then determinedas in the “simultaneous” and “forward” assays. In one embodiment, acombination of antibodies of the present invention specific for separateepitopes can be used to construct a sensitive three-siteimmunoradiometric assay.

Additionally, the exemplary antibodies can be utilized for T-celltyping, for isolating specific CPAA-bearing cells or fragments, forvaccine preparation, or the like. The antibodies may be used toquantitatively or qualitatively detect the CPAA in a sample or to detectpresence of cells that express the CPAA. This can be accomplished byimmunofluorescence techniques employing a fluorescently labeled antibody(see below) coupled with fluorescence microscopy, flow cytometric, orfluorometric detection. For diagnostic purposes, the antibodies mayeither be labeled or unlabeled. Unlabeled antibodies can be used incombination with other labeled antibodies (second antibodies) that arereactive with the humanized antibody, such as antibodies specific forhuman immunoglobulin constant regions. Alternatively, the antibodies canbe directly labeled. A wide variety of labels may be employed, such asradionuclides, fluors, enzymes, enzyme substrates, enzyme cofactors,enzyme inhibitors, ligands (particularly haptens), etc. Numerous typesof immunoassays, such as those discussed previously are available andare well known to those skilled in the art.

The antibodies useful in the present invention may be employedhistologically, as in immunofluorescence or immunoelectron microscopy,for in situ detection of the CPAA of the present invention. In situdetection may be accomplished by removing a histological specimen from apatient, and providing the labeled antibody of the present invention tosuch a specimen. The antibody (or fragment) may be provided by applyingor by overlaying the labeled antibody (or fragment) to a biologicalsample. Through the use of such a procedure, it is possible to determinenot only the presence of the CPAA but also its distribution on theexamined tissue. Using the present invention, those of ordinary skillwill readily perceive that any of wide variety of histological methods(such as staining procedures) can be modified in order to achieve suchin situ detection.

Importantly, the antibodies of the present invention may be helpful indiagnosing the invasiveness of certain types of colorectal andpancreatic cancer. More specifically, the antibody of the presentinvention may identify CPAA present in patients with slow cancers thatgrow over several years as opposed to aggressive cancers that progressmuch faster. Thus, the antibody of the present invention may provide animportant immunohistochemistry tool.

The antibodies of the present invention may be used on antibody arrays,highly suitable for measuring gene expression profiles includingpost-translational modification and also useful for detecting smallermolecules such as peptide hormones and carbohydrates. Several approacheshave recently been employed to determine the suitability and efficacy ofantibody arrays. In some instances, phage-displayed antibodies have beenused in preparing the arrays, and detection and analysis is done bySELDI (surface-enhanced laser desorption/ionization), or in ahigh-throughput format by filter-based enzyme-linked immunosorbent assay(EILSA). Other examples of detection systems include fluorescent tagsand nanoelectrodes, and for smaller arrays, surface plasmon resonanceand MALDI-TOF (matrix-assisted laser desorption ionization-time offlight) mass spectrometry. Proteome analysis can also be performed byfirst generating an array of bound antigens followed by antibody captureand detection with an affinity ligand such as Protein L or Protein Abound to a detection probe.

A third approach involves high-density gridding of bacteria containingantibody genes onto a filter followed by interaction with another filtercontaining an affinity ligand or the antigen attached with a detectionprobe such as ELISA. This method eliminates the need for liquidhandling, and parallel screens of tens of thousands of antibodiesagainst multiple antigens can be performed to identify ultimatelyproteins that are differentially expressed. A final method involves thepossibility of synthesizing antibodies directly on the chip usingcombinatorial chemistry. Current technology, however, somewhat strainedat synthesizing even the antigen-binding antibody domains that consistsof a minimum of 120 aminoacids, unless presynthesized polypeptidebuilding blocks are used to create an antibody framework followed by theaddition of individual amino acids.

Screening methods for determining anti-CPAA activities are also providedfor in the present invention. Specifically, as described further inExample 6, the antibody of the present invention is associated withantibody-dependent cellular cytotoxicity (ADCC) activity. Anti-CPAAcompounds that can be selected from the group consisting of antibodies,or fragments or portions thereof peptides, peptido mimetic compounds ororgano mimetic compounds that trigger death of CPAA-bearing cells invitro, in situ or in vivo are encompassed by the present invention.Screening methods which can be used to determine ADCC activity of ananti-CPAA compound can include in vitro or in vivo assays. Such in vitroassays can include a CPAA cytotoxicity assay, such as a radioimmunoassay, which determines a decrease in cell death by contact with CPAA,such as chimpanzee or human CPAA in isolated or recombinant form,wherein the concurrent presence of a CPAA neutralizing compound reducesthe degree or rate of cell death.

Diagnostic Kits

Kits can also be supplied for use with the subject antibodies in theprotection against or detection of a cellular activity or for thepresence of a selected antigen. Thus, an antibody of the presentinvention may be provided, usually in a lyophilized form in a container,either alone or in conjunction with additional antibodies specific forthe desired cell type. The antibodies, which may be conjugated to alabel or toxin, or unconjugated, are included in the kits with buffers,such as Tris, phosphate, carbonate, etc., stabilizers, biocides, inertproteins, e.g., serum albumin, or the like. Generally, these materialswill be present in less than 5% wt. based on the amount of activeantibody, and usually present in total amount of at least about 0.001%wt. based again on the antibody concentration. Frequently, it will bedesirable to include an inert extender or excipient to dilute the activeingredients, where the excipient may be present in from about 1% to 99%wt. of the total composition. Where a second antibody capable of bindingto the primary antibody is employed in an assay, this will usually bepresent in a separate vial. The second antibody is typically conjugatedto a label and formulated in an analogous manner with the antibodyformulations described above. The kit will generally also include a setof instructions for use.

Pharmaceutical Applications

The anti-CPAA antibodies or peptides of the present invention can beused for example in the treatment of carcinomas and related conditions.More specifically, the invention further provides for a pharmaceuticalcomposition comprising a pharmaceutically acceptable carrier or diluentand, as active ingredient, an antibody or peptide according to theinvention. The delivery component of the immunotoxin is a humanizedantibody according to the present invention. Intact immunoglobulins ortheir binding fragments, such as Fab, are also envisioned. Typically,the antibodies in the immunotoxins will be of the human IgA, IgM or IgGisotype, but other mammalian constant regions may be utilized asdesired. The composition may also comprise an immunotoxin according tothe invention. The humanized antibody, immunotoxin and pharmaceuticalcompositions thereof of this invention are useful for parenteraladministration, e.g., subcutaneously, intramuscularly or intravenously.

Anti-CPAA antibodies and/or peptides of the present invention can beadministered either as individual therapeutic agents or in combinationwith other therapeutic agents. They can be administered alone, but aregenerally administered with a pharmaceutical carrier selected on thebasis of the chosen route of administration and standard pharmaceuticalpractice.

For parenteral administration, anti-CPAA antibodies or peptides can beformulated as a solution, suspension, emulsion or lyophilized powder inassociation with a pharmaceutically acceptable parenteral vehicle. Forexample the vehicle may be a solution of the antibody or a cocktailthereof dissolved in an acceptable carrier, such as an aqueous carriersuch vehicles are water, saline, Ringer's solution, dextrose solution,or 5% human serum albumin, 0.4% saline, 0.3% glycine and the like.Liposomes and nonaqueous vehicles such as fixed oils can also be used.These solutions are sterile and generally free of particulate matter.These compositions may be sterilized by conventional, well knownsterilization techniques. The compositions may contain pharmaceuticallyacceptable auxiliary substances as required to approximate physiologicalconditions such as pH adjusting and buffering agents, toxicityadjustment agents and the like, for example sodium acetate, sodiumchloride, potassium chloride, calcium chloride, sodium lactate, etc. Theconcentration of antibody in these formulations can vary widely, forexample from less than about 0.5%, usually at or at least about 1% to asmuch as 15% or 20% by weight and will be selected primarily based onfluid volumes, viscosities, etc., in accordance with the particular modeof administration selected. The vehicle or lyophilized powder cancontain additives that maintain isotonicity (e.g., sodium chloride,mannitol) and chemical stability (e.g., buffers and preservatives). Theformulation is sterilized by commonly used techniques.

Thus, a typical pharmaceutical composition for intramuscular injectioncould be made up to contain 1 ml sterile buffered water, and 50 mg ofantibody. A typical composition for intravenous infusion could be madeup to contain 250 ml of sterile Ringer's solution, and 150 mg ofantibody. Actual methods for preparing parenterally administrablecompositions will be known or apparent to those skilled in the art andare described in more detail in, for example, REMINGTON'S PHARMA. SCI.(15th ed., Mack Pub. Co., Easton, Pa., 1980).

The antibodies of this invention can be lyophilized for storage andreconstituted in a suitable carrier prior to use. This technique hasbeen shown to be effective with conventional immune globulins. Anysuitable lyophilization and reconstitution techniques can be employed.It will be appreciated by those skilled in the art that lyophilizationand reconstitution can lead to varying degrees of antibody activity loss(e.g., with conventional immune globulins, IgM antibodies tend to havegreater activity loss than IgG antibodies) and that use levels may haveto be adjusted to compensate.

The compositions containing the present human-like antibodies or acocktail thereof can be administered for prevention of recurrence and/ortherapeutic treatments for existing disease. Suitable pharmaceuticalcarriers are described in the most recent edition of REMINGTON'SPHARMACEUTICAL SCIENCES, a standard reference text in this field of art.For example, a parenteral composition suitable for administration byinjection is prepared by dissolving 1.5% by weight of active ingredientin 0.9% sodium chloride solution. Anti-CPAA peptides and/or antibodiesof this invention can be adapted for therapeutic efficacy by virtue oftheir ability to mediate antibody-dependent cellular cytotoxicity(ADCC), and/or apoptosis, and/or complement-dependent cytotoxicity (CDC)against cells having CPAA associated with their surface. For theseactivities, either an endogenous source or an exogenous source ofeffector cells (for ADCC) or complement components (for CDC) can beutilized.

In therapeutic application, compositions are administered to a patientalready suffering from a disease, in an amount sufficient to cure or atleast partially arrest or alleviate the disease and its complications.An amount adequate to accomplish this is defined as a “therapeuticallyeffective dose.” Amounts effective for this use will depend upon theseverity of the malignancy and the general state of the patient's ownimmune system, but generally range from about 1 mg to about 200 mg ofantibody per dose, with dosages of from 5 mg to 25 mg per patient beingmore commonly used. It must be kept in mind that the materials of theinvention may generally be employed in serious disease states, oftenlife-threatening or potentially life-threatening situations. In suchcases, in view of the minimization of extraneous substances and thelower probability of “foreign substance” rejections which are achievedby the present human-like antibodies of this invention, it is possibleand may be felt desirable by the treating physician to administersubstantial excesses of these antibodies.

The dosage administered will, of course, vary depending upon knownfactors such as the pharmacodynamic characteristics of the particularagent, and its mode and route of administration; age, health, and weightof the recipient; nature and extent of symptoms kind of concurrenttreatment, frequency of treatment, and the effect desired. Usually adaily, weekly, or biweekly dosage of active ingredient can be about 100mg/m² to 250 mg/m² of body weight delivered over a 4 hour to 6 hourperiod.

As a non-limiting example, treatment of CPAA-related pathologies humansor animals can be provided as a daily, weekly, or biweekly dosage ofanti-CPAA peptides, monoclonal chimeric and/or murine antibodies of thepresent invention in a dosage range from 0.1 mg/kg to 100 mg/kg, perday, weekly, or biweekly.

Example antibodies for human therapeutic use are high affinity (thesemay also be high avidity) murine and chimeric antibodies, and fragments,regions and derivatives having potent in vivo anti-CPAA activity,according to the present invention.

Dosage forms (composition) suitable for internal administrationgenerally contain from about 0.1 mg to about 500 mg of active ingredientper unit. In these pharmaceutical compositions the active ingredientwill ordinarily be present in an amount of about 0.5%-95% by weightbased on the total weight of the composition.

Single or multiple administrations of the compositions can be carriedout with dose levels and pattern being selected by the treatingphysician. In any event, the pharmaceutical formulations should providea quantity of the antibody(ies) of this invention sufficient toeffectively treat the patient.

The antibodies can also be used as separately administered compositionsgiven in conjunction with chemotherapeutic or immunosuppressive agents.Typically, the agents will include cyclosporin A or a purine analog(e.g., methotrexate, 6-mercaptopurine, or the like), but numerousadditional agents (e.g., cyclophosphamide, prednisone, etc.) well-knownto those skilled in the art may also be utilized.

An antibody of the present invention may form part of an immunotoxin.Immunotoxins are characterized by two components and are useful forkilling selected cells in vitro or in vivo. One component is a cytotoxicagent which is usually fatal to a cell when attached or absorbed. Thesecond component, known as the “delivery vehicle”, provides a means fordelivering the toxic agent to a particular cell type, such as cellscomprising a carcinoma. The two components are commonly chemicallybonded together by any of a variety of well-known chemical procedures.For example, when the cytotoxic agent is a protein and the secondcomponent is an intact immunoglobulin, the linkage may be by way ofheterobifunctional cross-linkers, e.g., SPDP, carbodiimide,glutaraldehyde, or the like. Production of various immunotoxins iswell-known with the art, and can be found, for example in Thorpe et al.,Monoclonal Antibody—Toxin Conjugates. Aiming the Magic Bullet, inMONOCLONAL ANTIBODIES IN CLIN. MED. 168-90 (Acad. Press, 1982).

A variety of cytotoxic agents are suitable for use in immunotoxins.Cytotoxic drugs interfere with critical cellular processes includingDNA, RNA, and protein synthesis. Cytotoxic agents can includeradionuclides, such as include ²¹²Bi, ¹³¹I, ¹⁸⁸Re, and ⁹⁰Y; a number ofchemotherapeutic drugs, such as vindesine, methotrexate, adriamycin, andcisplatin; and cytotoxic proteins such as ribosomal inhibiting proteinslike pokeweed antiviral protein, Pseudomonas exotoxin A, ricin,diphtheria toxin, ricin A chain, etc., or an agent active at the cellsurface, such as the phospholipase enzymes (e.g., phospholipase C). Seegenerally, Olsnes & Phil, Chimeric Toxins, 25 Pharmac. Ther. 335-81(1982); MONOCLONAL ANTIBODIES FOR CANCER DETECTION & THERAPY, 159-79,224-66 (Baldwin & Byers eds., Acad. Press, 1985).

The antibodies or peptides and derivatives can be used therapeuticallyas immunoconjugates. See Dillman, 111 Ann. Internal Med. 592-603 (1989).Such antibodies or polypeptides can be coupled to cytotoxic proteins,including, but not limited to ricin-A, Pseudomonas toxin and Diphtheriatoxin. Toxins conjugated to antibodies or other ligands or peptides arewell known in the art. See, e.g., Olsnes et al., 10 Immunol. Today291-95 (1989). Plant and bacterial toxins typically kill cells bydisrupting the protein synthetic machinery. Cytotoxic drugs that can beconjugated to anti-CPAA peptides and/or antibodies and subsequently usedfor in vivo therapy include, but are not limited to, daunorubicin,doxorubicin, methotrexate, and Mitomycin C. For a description of theseclasses of drugs which are well known in the art, and their mechanismsof action, see Goodman & Gilman's PHARMACOLOGICAL BASIS OF THERAPEUTICS(8th Ed., Macmillan Pub. Co., 1990). Additionally, the antibody of thepresent invention may be delivered in combination with chemotherapeuticagents such as oxaliplatin, irinotecan, topotecan, leucovorin,carmustine, vincristine, fluorouracil, streptozocin, and gemcitabine.Combinations of other antibodies and such compounds have been used inadvanced colorectal cancer patients. See, e.g., U.S. Patent ApplicationPub. No. 20020187144.

Anti-CPAA antibodies and/or peptides of this invention can beadvantageously utilized in combination with other monoclonal or murineand chimeric antibodies, fragments and regions, or with lymphokines orhemopoietic growth factors, etc., which serve to increase the number oractivity of effector cells which interact with the antibodies. Forexample, the antibody of the present invention may be co-administeredwith human monoclonal antibodies reactive with other markers on cellsresponsible for the disease. For example, suitable T-cell markers caninclude those grouped into the so-called “Clusters of Differentiation”as named by the First International Leukocyte Differentiation Workshop,in LEUKOCYTE TYPING (Bernard et al., eds., Springer-Verlag, NY, 1984).

The 16C3 Antigen

The antigen to which the 16C3 antibody binds appears to be expressed insome, but not all, cultured human tumor cell lines, in some but not allnormal human embryonic gut tissues, and in most colon and pancreas tumortissues. As detailed in the Examples, below, upon western blotting ofvarious tumor samples, the 16C3 antibody recognized both a 200 kDapeptide species and a ˜110 kDa peptide species. The intensity of thestaining, which may reflect the amount of each protein species from aparticular sample, appears to differ between colorectal tumor samplesand pancreatic tumor samples. The 16C3 antigen appears to be expressedin most colon and pancreas tumor tissues, and may have an oncofetalorigin. The 16C3 antigen is expressed on the cell surface and is aglycoprotein. There may be two related species of the same antigen thatthe 16C3 antibody recognizes, and the relative amount of each speciesdiffers between colon and pancreas tumor tissues.

Binding of the 16C3 antigen by western blotting is not disrupted bytreatment with either detergents such as SDS and NP-40, or reducingagents such as dithiothreitol and 2-mercaptoethanol; suggesting thatbinding to the specific epitope is not conformation-dependent, and that16C3 antibody may recognize a linear epitope on the CPAA. Furthermore,the 16C3 epitope is unaffected by treatment with V8 protease, trypsin orPNGase-F, although the overall migration of the immunoreactive antigenon SDS-PAGE was changed, reflecting modification by proteolysis anddeglycosylation. Treatment with sodium hydroxide in a beta-eliminationchemical reaction appeared to result in a loss of immunoreactivity,suggesting that the 16C3 epitope may involve an O-linked glycosylationmodification, or lie adjacent to an O-linked glycosylation residue.Further studies define the precise epitope to which the 16C3 antibodyreacts.

The characterization of the 16C3 epitope may also lead to theidentification of the gene(s) for the CPAA, such as that it may providea target for translation antagonists or other means of blockingexpression, or an understanding of the CPAA activity such that it maybecome a target for antagonists, particularly small molecules orantibodies, which block functional activity (such as, for example,binding of the receptor by its cognate ligand(s); transport function;signaling function).

Cancer Vaccine

Another aspect of the present invention provides for a cancer vaccine.By “vaccine” is meant an agent used to stimulate the immune system of aliving organism. In this regard, the immune response may provide forprophylaxis or may provide for a positive effect in a diseased organismby, for example, alleviating an existing condition. Specifically, acancer vaccine is meant to therapeutically treat existing malignancyand/or to prevent the progression or metastasis of an existingmalignancy.

That specific active immunotherapy can be achieved usingtumor-associated antigens is widely known. Indeed, the initial,semi-purified antigenic preparations used to derive the monoclonalantibody that has allowed the further invention presented herein wereshown to provide specific, active, long-lasting protective immunity inhumans. Hollinshead et al., 1985. At that time, patients had undergonetumor resection and were then vaccinated with antigenic material derivedfrom tumor membranes in the amount of 200 μg, 300 μg, or 500 μg in 0.2ml dispersions mixed with an additional 0.2 ml Freund's adjuvant.Dosages of 300 μg given monthly for three months were shown to be safe.

With the recombinant antibodies described herein, it is now possible todefine a highly purified antigen or epitope peptides of CPAA that isfurther suitable for a vaccine against these cancers. For example, 16C3may be used to bind to tissue or cell samples from which the CPAAprotein and its corresponding amino acid sequence may be identified byany number of known techniques. The epitope may be mapped further, andthe molecular nature determined with exquisite detail. See, e.g.,Baerga-Oritz et al., 11 Protein Sci. 1300-08 (2002); Jemmerson, &Paterson, 4 BioTechniques 18-31 (1986).

An alternative technique to identify effective antigenic peptidesentails using the 16C3 antibody or peptide to screen an expressionlibrary (such as a phage display library) for mimetic proteins, ormimotopes, that are recognized by the antibody. This technique has beenused to identify antigenic peptides that have raised protective immuneresponses in vivo. See Beenhouwer et al., 169 J. Immunol. 6992-99(2002); see also U.S. Pat. No. 5,837,550; Visvanathan et al., 48Arthritis & Rheumatism, 737-45 (2003); Sato et al., 371 Biochem. J.603-08 (2003). Note that this technique has been used to identifyprotein mimetics of carbohydrate and glycoprotein antigens, the proteinversions found to be more immunogenic than the natural carbohydratecounterparts. Indeed, mimetics may be isolated that are advantageousover known antigens because of factors including production capacity,safety, half-life, or other issues.

The CPAA immunogenic protein may be prepared and delivered, for example,as either a subcutaneous or a mucosal vaccine alone, or associated withan adjuvant or carrier or as part of an adjuvant or protein conjugate.Delivery by liposomes microparticles, virus-like particles, DNAvaccines, live recombinant vectors such as S. typhimurium, and possiblyISCOMs are envisioned. All of these systems are well-known by those ofordinary skill in the art, and may be practiced without undueexperimentation. See, e.g., Michalek et al., in MUCOSAL IMMUNOLOGY(Mestecky et al., eds., Elsevier, 2005).

Additionally, the CPAA peptide may be genetically or chemicallyconjugated to a toxoid carrier, such as cholera, entero-, or ricintoxoid. See, e.g., U.S. Pat. No. 6,846,488. Another advantageous proteincarrier derived from bacterium is the PorB protein carrier. See e.g.,U.S. Pat. No. 6,613,336. Another promising protein-based mucosaladjuvant is the flagellin protein from S. typhimurium. In an embodimentof the invention, the CPAA protein is co-administered with the flagellinprotein (FljB) via, for example, the mucosal intranasal route. Anadvantageous protein platform comprising duck hepatitis core antigen isalso presented in U.S. Patent Application Pub. No. 20040219164.

The CPAA of the present invention may also be delivered as a DNA vaccinefor in vivo expression of the immunogenic construct. For example,cationic microparticles may be used to deliver the DNA expressioncassette in intranasal vaccination. Such systems have induced an immuneresponse following, for example, intranasal delivery of vaccinecomprising DNA encoding the HIV-1 gag protein. Michalek et al., 2005. Inan embodiment of the present invention, the CPAA immunogenic peptide isdelivered via a DNA expression cassette which is subsequently expressedin vivo.

Additionally, the immunogenic preparation may be used to “charge” donorderived dendritic cells ex vivo, which are then returned to the patientwhere they home to the lymphoid organs and mount an effective immuneresponse. See, e.g., Liau et al., 9(6) Neurosurg. Focus, e8 (2000);Baar, 4(2) Oncologist 140-44 (1999). This vaccine approach has been inhuman trials for treating, for example, melanoma and brain cancer. Moreinformation may be found on-line at, for example, the NationalInstitutes of Health's web site for clinical trials. Alternatively, aDNA vaccine as described above may be delivered via skin patch to thecells of Langerhans, which then mature to dendritic cells and home tothe lypmphoid organs. U.S. Pat. No. 6,420,176.

Delivery of the immunogenic compositions of the present invention may beby parenteral, subcutaneous, or intramuscular injection, intravenousinjection, intestinal, intradermal, intubation, or nasal, oral or rectalvaccination. The vaccine may also be delivered topically, includingintranasal, upon the palatine tonsil, or delivery to the salivaryglands. In other words, a vaccine contemplated by the present inventionmay be administered to the patient by any known or standard techniques.

The invention will now be described further by non-limiting examples.

EXAMPLES Example 1 Preparation of Pancreatic and ColorectalCarcinoma-Associated Antigen (CPAA) from Human Tumor Specimens

An immunogenic tumor associated antigen preparation was obtained frompooled colorectal carcinoma membranes according to the method describedby Hollinshead et al., 56 Cancer 480 (1985); U.S. Pat. No. 5,688,657.This antigenic material was purified to the extent that the membranefractions were free of HL-A antigens and were separated from much of thenon-immunogenic glycoprotein fractions.

Tumor cell suspensions in saline were prepared from fresh operating roomspecimens. Single cell suspensions, obtained by mincing solid tumors,were centrifuged for 10 min. at 400× gravity and the supernatant wasretained. The cell pellet was resuspended in phosphate buffered saline(PBS) and re-centrifuged. The membrane material was examined by electronmicroscopy to assure that only membrane material (and no intact cells)was present, and the protein content was measured by the Lowry method.The membrane material was next subjected to sequential low frequencysonication and resuspended as a soluble pool of membrane proteins. Thesoluble sonicates were separated by gel filtration on Sephadex-6200.Fractions of 2 ml were collected and the absorbance profile at 220 nmand 280 nm was recorded. Fractions comprising individual protein peakswere pooled, and the pools were concentrated by Diaflo ultrafiltration.Sephadex-G200 fractions IB and IIA, as defined by Hollinshead et al.,1985, were further purified by gradient polyacrylamide gelelectrophoresis (PAGE). The fractions were tested for their ability toelicit positive delayed cutaneous hypersensitivity reactions in patientswith colorectal carcinoma. Those fractions with immunogenic activitywere said to contain colorectal carcinoma-associated antigens and wereemployed as immunogens and screening agents in the preparation ofmonoclonal antibodies.

By gradient PAGE, a double-banded antigen distinct from that ofcarcinoembryonic antigen was identified and isolated. Gold et al., 122J. Exp. Med. 467-81 (1965); Hollinshead et al., 1985; Hollinshead etal., 1(7658) Lancet 1191-1195 (1970); Hollinshead et al., 177 Science887-89 (1972). The bands comprising this antigen migrated 6.3 cm and 6.6cm distant from tracking dye. Biochemical analysis of the antigenindicated that this protein was a glycoprotein. The molecular weight ofthe antigen was estimated based on the electrophoretic mobility oftransferrin (6.4 cm-6.5 cm), one isolate has a molecular weight of 76.5kDa. The semi-purified antigens were studied in detail, includingassessments of serum antibodies, cell-mediated immunity, and patientsurvival. Hollinshead et al., Abstract, Ann. Meeting Am. Soc'y Clin.Oncol., Washington, D.C. (1990); Hollinhead et al., 56 Proc. 6th Int'lConf. Adjuvant Therapy Cancer (Salmon, ed., W.B. Saunders Inc, Phila.,PA, 1990). Additional studies were performed to evaluate the usage ofcombination immuno-chemotherapies. Hollinshead et al., 1990a;Hollinshead et al., 1990b; Hollinshead, 7 Sein. Surg. Oncol. 199-210(1991); Hollinshead et al., 10(1) J. Exper. Clin. Cancer 43-53 (1991);see also Hollinshead & Herberman, Proc. 2nd Int'l Symp. Cancer Detection& Prevent. 616-20, (Bologna, Italy, 1973); Hollinshead, Experience withcombo. immuno-chemotherapy of colon cancer: steps pertinent tosuccessful therapy based upon dosage & timing of admin. of 5-FU. NIHWorkshop on Levamasole: Mechanism of anti-tumor action (Bethesda, Md.,Jun. 11, 1990); Hollinshead, 7 Semin. Surg. Oncol., 199-210 (1991);Hollinshead, 8(153) Clin. Exper. Metastasis 89 (1990).

Example 2 Immunization and Preparation of Hybridomas

Monoclonal antibodies against human pancreatic and colorectalcarcinoma-associated antigens were obtained by the production andcloning of hybrids resulting from the fusion of mouse myeloma cellsSp2/0-Ag14 with spleen cells from BALB/c mice which had been immunizedwith the CPAA described above. Hybrid clones were established andreacted strongly with the CPAA and with a colon carcinoma cell line(LS174T) when assayed by ELISA.

Immunization and Cell Fusion: BALB/c mice were immunized byintraperitoneal injection of 100 μg of the CPAA emulsified in completeFreund's adjuvant. The CPAA was prepared as described by Hollinshead inclinical trials. Four weeks later, a second immunization with 50 μg ofCPAA emulsified in incomplete Freund's adjuvant was performed. Fourteendays later the mice received an intraperitoneal booster injection of 50μg of CPAA emulsified in incomplete Freund's adjuvant. Mice weresacrificed three days later and a single cell splenocyte suspension wasprepared. Cell fusion was performed by incubation of 5e7 mouse spleencells with 10e7 sP2/0-Ag14 myeloma cells in 40% polyethylene glycol(MW=1500).

Screening of Hybridoma Clones: An enzyme-linked immunosorbent assay(ELISA) was used to detect hybridoma clones producing antibodiesspecific for the PCAA. Colon tumor cell membrane extract (10 ng/well ofLS174T or HT-29) served as a surrogate source of colon cancer antigensand was immobilized on polystyrene microplates. Antibodies present inthe test supernatants were allowed to bind to the immobilized antigensfor one hour. The presence of the bound murine mAbs was detected withphosphatase-conjugated secondary antibodies, specific for mouseimmunoglobulin. Wells were washed and then the chromogenic substrate foralkaline phosphatase (pNPP) was added. Wells showing color reactionsyielding absorbances greater or equal to 0.500 units were scored aspositive. Negative controls gave values of 0.01 to 0.09 absorbanceunits. Hybridoma wells scoring as positive by ELISA were selected forexpansion and repeating the cell cloning procedure by the limitingdilution cloning method. Selection of positive mAb producing hybridomacells was determined by ELISA. Positive monoclonal cells were expandedin culture and aliquots of the cells were frozen under liquid nitrogenfor long term storage.

Example 3 Isotype of the 16C3 mAb

Murine immunoglobulins are expressed from separate genes that encode theheavy chain (55 kD) and the light chain (25 kD-29 kD). There are fourheavy chains of the IgG subclass (IgG1, IgG2a, IgG2b, IgG3) and twolight chains (Kappa, Lambda) that can rearrange to yield the repertoireof murine immunoglobulins.

The isotype of the 16C3 mAb was determined using the SouthernBiotechnology, Inc. mouse isotyping kit. The 16C3 mAb was determined tobe an IgG1 heavy chain and a Kappa light chain.

Example 4 Unique DNA Sequences Encode the 16C3 Antibody

The linear amino acid sequence of a mAb identifies its uniqueness, incomparison to the known sequences of all other mAbs described. Thelinear amino acid sequence can be determined by first determining thelinear sequence of the DNA that encodes the polypeptide molecule. TheDNA sequence that encodes the 16C3 mAb was determined and the openreading frame was translated into the amino acid sequence using theuniversal mammalian codon usage table, thus describing the linearsequence identity of the 16C3 molecule.

Oligonucleotide primers used for the murine IgG1 heavy chain kappa lightchain cloning derived from the publication Rapid cloning of anyrearranged mouse immunoglobulin variable genes, Dattamajumdar et al., 43Immunogenetics 141-51 (1996).

Isolation of the nucleic acid of 16C3: Ribonucleic acid (RNA) wasisolated from the 16C3-producing hybridoma cells using the KNeasy-Midikit (catalog #74104, Qiagen, Valencia, Calif.) as described by themanufacturer. Four million cells were centrifuged in a conical tube, andthe cells were lysed to release the nuclear and cytosolic nucleic acidsincluding the RNA. The RNA was then purified from the lysate using theRNeasy spin columns. Finally, the RNA was eluted with water and analyzedfor yield and purity by absorbance at 260 nm and 280 nm using aspectrophotometer. The isolated RNA was stored at −80° C.

Preparation of the cDNA: The RNA (2 pg) was first reverse-transcribed tocDNA using a deoxynucleotide triphosphate dNTP mixture (dATP, dCTP,dGTP, dTTP), cDNA systhesis buffer, RNase inhibitor, reversetranscriptase enzyme, and oligo(dT)₂₀. The cDNA synthesis reaction wasperformed according to the manufacturer's instructions in Invitrogen'sSuperscript III kit (catalog #18080-051). The target cDNA (mouse IgG1heavy chain and Kappa light chain) were amplified for sequencingpurposes by the polymerase chain reaction (PCR) following theinstructions recommended by the Accuprime Pfx DNA polymerase kit fromInvitrogen (catalog #12344-024) using the forward and reverse primersdescribed above for both the heavy chain and light chain. The mixturewas subjected to 940C for 2 min., followed by thirty cycles of: 15 sec.at 94° C., 30 sec. at 58° C., 30 sec. at 68° C., which was then followedby 10 min. at 68° C. The amplified heavy and light chain DNA fragmentswere then electrophoresed on a 4% NuSieve 3:1 plus agarose gel(Lonza-Rockland, catalog #54925). The target DNA bands were excised fromthe gel and then purified from the agarose using QIAquick gel extractionkit (catalog #28704, Qiagen).

DNA sequencing and analysis: Amplified target DNA representing thevariable regions of the heavy chain and light chain of 16C3 antibody wasTOPO cloned for sequencing according to the manufacturers instructions(Invitrogen catalog HK4530-20). Several TOPO clones were selected andsubjected to DNA sequencing. Full-length sequences for the 16C3 antibodywere obtained using the 5′/3′ RACE kit according to the manufacturer'sinstructions (Roche Applied Sci., catalog #03-353). The DNA sequencesobtained were translated in three reading frames and the frame withoutstop codons and that aligned homologously with other murine heavy andlight chains was determined to be the genuine reading frame. The DNAsequence was used as the query sequence to search the National Centerfor Biotechnology Information (NCBI) database (All GenBank+RefSeqNucleotides+EMBL+DDBJ+PDB sequences). The BLAST search returned up tofifteen database entries with nucleotide sequence similarity to thequery sequence of 16C3. None of the DNA sequences were identical to the16C3 DNA sequence, demonstrating the uniqueness of the 16C3 mAbdescribed herein.

Example 5 Uniqueness of the 16C3 Antibody Confirmed by BLAST DatabaseSearch

The sequences of the 16C3 mAb were subjected to BLAST searching (BasicLocal Alignment Search Tool) against the protein and nucleic aciddatabase at the National Center for Biotechnology Information CBI), partof the National Institutes of Health's National Library of Medicine.Examination of the similar sequences found by this BLAST search witheither 16C3 mAb heavy chain or 16C3 mAb light chain query sequencesindicated that both the 16C3 mAb heavy chain and light chain variableregions are unique sequences. Thus, the 16C3 mAb heavy and light chainsequences represent a novel and unique antibody molecule.

Example 6 Specific Cell Binding of 16C3 mAb

The 16C3 mAb produced by the hybridoma was purified by affinitychromatography using protein L-agarose matrix. The purified 16C3 mAbwascharacterized by indirect immunofluorescence, using various tumor cellsas identified in Table 1, below. All of the tumor cell lines wereobtained from the ATCC. Cells were incubated with purified 16C3 mAbdiluted in phosphate buffered saline (PBS) for 1 hr at 4° C. The cellswere washed and incubated with a fluorescein-labelled goat anti-mousemAb. The cells were then washed three times with PBS and examined byflow cytometry using a Becton-Dickinson FACSCaliburm™ and CellQuestanalysis software. The results appear in Table 1 (FACS data). The datademonstrate the specific binding of 16C3 mAb to colorectal andpancreatic tumor cell lines, but not to prostate or squamous tumor celllines.

TABLE 1 16C3 mAb FACS data: binding to tumor cell lines % Cell Staining(mfi) Tumor Cell Line FITC-Ab only Rockland 16C3-E12 LS174T Colorectal0.94 (15) 40.56 (59)  HT-29 Colorectal 0.84 (10) 90.99 (78)  CFPAC-1Pancreatic 0.83 (14) 96.19 (323) AsPC-1 Pancreatic 2.68 (30) 69.90 (36) 22Rv-1 Prostate 2.74 (61) 1.62 (30) PC-3 Prostate 0.28 (20) 2.44 (22)H226 Squamous 0.90 (18) 0.62 (14) SiHa Squamous 1.07 (19) 1.08 (20)

Example 6 ADCC Activity of 16C3 Demonstrating Anti-Tumor Cytotoxicity

A therapeutically useful mAb, specific for an immunogenic tumor antigen,may have at least one of the following properties. (a) high tumor tissuespecificity, (b) absence of cross-reactivity to normal human tissue, and(c) a biological activity associated with destruction of tumors, such asantibody-dependent cellular cytotoxicity (ADCC). The ADCC activity ofthe 16C3 mAb was tested on colon SW1463 and pancreatic CFPAC-1 andAsPC-1 carcinoma lines as target cells. The melanoma cell line, SK-MEL,served as a specificity control. ADCC was assayed using a conventional4-hour ¹¹¹In release assay using normal human PBMC as effector cells,and the results are shown as the percent isotope release (% lysis) inTable 2 (ADCC data). Compared to the negative control antibody, UPC-10,the data indicate a modest killing activity of the murine IgG1 antibody,but, importantly, the killing activity is apparently specific for colonand pancreatic tumor lines. The killing activity of an antibody mayincrease with humanized or chimerized antibody having human frameworksequences that include the Fc region, which interacts with humaneffector cells in this ADCC assay.

TABLE 2 ADCC assay with murine 16C3 mAb % Specific ADCC ActivityEffector:Target (±SEM) Tumor Target Ratio 16C3 mAb UPC-10 SW1463 50:1 4.1 ± 0.4 1.6 ± 0.3 (colorectal adeno) 25:1  5.2 ± 0.3 −0.2 ± 0.1  CFPAC-1 50:1 11.1 ± 2.7 0.2 ± 0.6 (pancreas adeno) 25:1  1.4 ± 0.7 −0.2± 0.3   AsPC-1 50:1 16.1 ± 0.8 0.9 ± 0.3 (pancreas adeno) 25:1 10.6 ±1.0 0.4 ± 0.2 SK-MEL 50:1 −3.0 ± 0.2 −0.5 ± 0.2   (melanoma) 25:1 −3.3 ±0.1 −2.0 ± 0.2   ¹¹¹In labeled target cells, antibodies used at 5μg/well, IL-2 activated human PBMC used as effector cells, 4 hrincubation at 37° C. before harvest.

Example 7 SDS Polyacrylamide Gel Electrophoresis Analysis of the 16C3Antibody

The native configuration of murine immunoglobulin gamma (IgG1) iscomprised of four polypeptides, with two polypeptides each of a heavychain and a light chain. One heavy chain (55 kDa) is associated with onelight chain (25 kDa-29 kDa) and this dimer is linked to an identicaldimer through disulfide bonding to complete the functional tetramericmacromolecule. The IgG molecule can be dissociated into its componentheavy and light chains and separated by size on polyacrylamide gelmatrix in the presence of denaturing reagent (SDS, sodium dodecylsulfate) and an agent to reduce the disulfide bridge that links the twoheterodimers (DTT, dithiothreitol). Gel electrophoresis is a commonanalytical method used to define the molecular mass of proteinaceousmaterials, such as antibodies.

Purified 16C3 mAb was subjected to analysis by SDS polyacrylamide gelelectrophoresis in the presence of reducing agent (DTT). Five microgramsof purified 16C3 mAb was mixed with DTT and 4× samples buffer containingSDS, glycerol, and bromophenol blue dye. The mixture was heated to 95°C. for 2 min., cooled on ice, then loaded onto an SDS gradientpolyacrylamide gel (4%-20% gradient) and subjected to an electriccurrent to separate the denatured molecular species in the 16C3 mAbpreparation. Following electrophoresis, the gel was stained withCoomassie Blue dye to visualize the proteins on the gel, destained withwater, and dried between pourous plastic sheets. The data demonstratetwo protein bands of molecular mass 55 kDa, representing the heavychain; and 28 kDa, representing the light chain molecular species. Thesedata show that the purified material correspond to the known molecularsizes for murine IgG1 heavy and light chain proteins.

Example 8 Immunohistochemical Staining with 16C3 mAb and Human MalignantTissues

The specificity of antigen binding displayed by the 16C3 mAb wasmeasured by immunohistochemical staining of various human tissuesamples, both cancer and normal specimens. Paraffin and fresh frozenhuman tissue samples were stained with purified mouse 16C3 Mab (IgG1),at 5&g/mL, then detected using a peroxidase-conjugated anti-mouse IgGsecondary antibody. The intensity of staining is indicated in Table 3,reflecting a 0-4 rating system: 0 indicating no cross-reactivity; 4indicating very high cross-reactivity to the antigen or high expressionof the antigen in a given specimen.

TABLE 3 Immunohistochemical staining indicating 16C3 specificity. numberSample preparation method positive/total stain intensity Paraffin-Coloncancer 31/33 3+, 4+ Paraffin-Colon normal  0/18 0 Paraffin-Pancreascancer 17/18 3+, 4+ Paraffin-Pancreas normal 0/8 0 Paraffin-Colon cancer2/2 2+, 3+ Paraffin-Lung-adeno cancer 2/2 1+, 2+ Paraffin-Mucinous Ovarycancer 2/2 2+, 3+ Paraffin-Liver-Cholagiocarcinoma 2/2   1+Paraffin-Stomach cancer 2/2 3+, 4+ Paraffin-Uterus-Cervix cancer 2/2 1+,2+ Paraffin-Uterus-Endometrial cancer 0/2 0 Paraffin-Prostate cancer 0/40 0 Paraffin-Serous Ovary cancer 0/2 0 Paraffin-Bladder-Transitional0/2 0 cell cancer Paraffin-Kidney cancer 0/2 0 Paraffin-Lung-squamouscancer 0/2 0 Paraffin-Kidney cancer 0/2 0 Paraffin-Lung-squamous cancer0/2 0 Paraffin-Esophagus-Squamous 0/2 0 Paraffin-Liver-Hepatoma 0/2 0Paraffin-Thyroid, papillary 0/2 0 Paraffin-Thyroid, follicular 0/2 0Paraffin-Breast, ductal 0/2 0 Paraffin-Skin-Squamous 0/2 0Paraffin-Stomach, signet ring 0/2 0 Paraffin-various normal  0/54 0Fresh-frozen-Colon cancer 2/3 2+, 3+ Fresh-frozen-Colon normal 0/2 0Fresh-frozen-Pancreas cancer 2/3 2+, 3+ Fresh-frozen-Pancreas normal 0/20

Considered collectively, these data demonstrate over 90% bindingspecificity to colon (35/38) and pancreas (19/21) cancer tissues,whereas there was no cross-reactivity to any normal human tissues (0 outof 58 tested). There were also some cross-reactivity to other tumortypes including lung adenocarcinoma (2/2), mucinous ovarian cancer(2/2), liver cholangiocarcinoma (2/2), stomach cancer (2/2), anduterine-cervix cancer (2/2). These data may indicate a generalcross-reactivity with an antigen present on adenocarcinomas.

Example 9 Humanization of Murine 16C3 Monoclonal Antibody

To improve usefulness as a therapeutic drug to treat human malignancy, amouse monoclonal antibody may be converted to a chimerized or humanizedantibody, such that the drug may be administered both repeatedly andwith lower toxicity. It is known in the art that administration of amurine protein may sometimes result in massive immune and toxicresponses to the foreign protein. Hence, a humanized antibody may provemore efficacious for human therapies.

The humanization of mouse antibodies for therapeutic applications iswell known, and several techniques for making humanized mAbs arediscussed above. For example, according to the Frankenstein approach,human framework regions are identified as having substantial sequencehomology to each framework region of the relevant non-human antibody,and CDRs of the non-human antibody are grafted onto the composite of thedifferent human framework regions. See U.S. Patent Appl. Pub. No.20060088522. A related method also useful for preparation of antibodiesof the invention is described in U.S. Patent Appl. Pub. No. 20030040606.

Five different alternative sequences for converting the murine 16C3antibody to a humanized, therapeutically useful antibody were designed.The designs were based upon structural information about known murineand human antibody sequences. Each of these variable regions were fusedin-frame to a known human IgG1 framework antibody that has been commonlyused for other therapeutic antibodies such as CC49 and CC83. The geneswere chemically synthesized, the sequences were verified, and then theheavy and light chain gene inserts were cloned into a mammalianexpression plasmid under the control of the CMV promoter. It should benoted that the variant light chain designs for VEN16C3 and CDR16C3 areidentical so only one light chain gene specified by this sequence designwas synthesized. The plasmids encoding each heavy chain and light chainwere co-transfected into human 293T cells using standardlipofectamine-based methods and reagents.

The humanized mAb sequences shown in FIG. 6 (light chain sequences) andFIG. 7 (heavy chain sequences) represent potential therapeutic forms ofthe 16C3 antibody for use in treating human malignancy. Human germlineIgG sequences were used for the framework sequences. The abbreviationsare as follows: 16C3 is the murine antibody sequence, ven16C3 has beenveneered with human framework sequences, cdr16C3 has been remodeled withhuman CDR amino acids, abb16C3 represents abbreviated CDR grafting,sdr16C3 represents site determining amino acid changes, and fra16C3represents a “Frankenstein” approach to remodeling the variable regionby using a combination of various “pieces” of human variable regions.Numbering is Kabat numbering.

Example 10 Specific Cell Binding of Recombinant Mouse 16C3 mAb

The mouse 16C3 mAb produced by the hybridoma was purified by affinitychromatography using protein L-agarose matrix. Recombinant mouse 16C3mAb produced by Chinese hamster ovary cells (CHO) was purified byaffinity chromatography using protein A-Sepharose matrix. The purified16C3 preparations, or transfected cell supernatants, were characterizedby indirect immunofluorescence using human colorectal LS174T orpancreatic AsPC-1 tumor cells as shown below. Cells were incubated withpurified 16C3 (mouse or humanized) diluted in phosphate buffered saline(PBS) for 1 hour at 4° C. The cells were washed and incubated with afluorescein-labelled goat anti-mouse immunoglobulin antibody. The cellswere then washed with PBS and examined by flow cytometry using aBecton-Dickinson FACScalibur and CellQuest analysis software. The datademonstrate very similar binding of both hybridoma-derived andrecombinant CHO-derived mouse 16C3 to colorectal and pancreatic tumorcell lines, but not to prostate or squamous tumor cell lines.

TABLE 4 Hybridoma vs. Recombinant mouse 16C3 FACS data, binding to tumorlines Antibody Sample % LS174T tumor cell binding (MFI) Goat anti-MouseIgG-FITC control 1.63 (43) Purified m16C3, from H12 hyb. 525, 2 ug 43.11(118) Purified m16C3, from H12 hyb. 712, 2 ug 43.56 (113) Purifiedm16C3, from H12 hyb. 713, 2 ug 37.79 (110) Purified m16C3, from H12 hyb.840, 2 ug 35.72 (92)  Purified m16C3, from rec-CHO, 1019, 2 ug 41.45(112) Purified m16C3, from rec-CHO, 1115, 2 ug 42.51 (114) Purifiedm16C3, from rec-CHO, 1220, 2 ug 37.46 (110) % AsPC-1 Cells Stained (mfi)Goat anti-Mouse FITC control 1.01 (8)  mouse 16C3-hybridoma control,712, 1 ug 89.50 (138) mouse 16C3-hybridoma control, 713, 5 uL 81.42(34)  Rec-CHO m16C3 supe #1 82.66 (28)  Rec-CHO m16C3 supe #2 83.86(27)  Rec-CHO m16C3 supe #3 79.50 (18)  Rec-CHO m16C3 supe #4 83.08(25) 

Example 11 Immunohistochemical Staining of Human Tissues UsingRecombinant Mouse 16C3 Antibody

The specificity of antigen binding displayed by the 16C3 antibody wasmeasured by immunohistochemical staining of various human tissuesamples, both cancer and normal specimens. Tissue microarrays,paraffin-embedded tissues, and fresh frozen human tissue samples werestained with purified mouse 16C3 antibody (IgG1), at 5 ug/mL, thendetected using a peroxidase-conjugated anti-mouse IgG secondaryantibody. The intensity of staining is indicated using a 0-4 ratingsystem, with 0 indicating no cross-reactivity and 4 indicating very highcross-reactivity to the antigen or high expression of the antigen in agiven specimen. Both the hybridoma-derived 16C3 antibody and therecombinant 16C3 antibody were tested. The results are summarized in theTables 5 and 6, below:

TABLE5 Staining with mouse 16C3 purified from hybridoma cells. Numberpositive/number Staining Human Tissue Sample stained intensity Coloncancer 17/18 +3 to +4 Colon cancer mets 18/18 +3 to +4 Pancreas cancer28/33 +1 to +3 Various other cancer tissues  8/18 +1 to +3 Normal colon,pancreas, and  0/74 other tissues

TABLE 6 Staining with recombinant mouse 16C3 purified from CHO cells.Number positive/number Human Tissue Sample stained Staining intensityColon cancer 45/45 +2 to +3 Pancreas cancer 24/30 +1 to +3 Various othercancer tissues 116/191 weak to +4 Normal colon, pancreas, 22/50 weak to+2 and other tissues

Considered collectively, these data demonstrate over 95% bindingspecificity to colon cancers (80/81), approximately 80-85% bindingspecificity to pancreas cancers (52/63), and 40-60% binding specificityto other types of cancer (predominantly adenocarcinomas). There was somecross-reactivity to a small subset of normal tissues, most notably lungand ovarian tissues, and the overall cross-reactivity was approximately44% (22/50). Interestingly, all normal human tissues that cross-reactedto the 16C3 antibody were performed with the recombinant producedantibody, (no cross-reactivity to normal human tissues observed with thehybridoma-produced 16C3), suggesting that the cross-reactivity may berelated to an artifact of the CHO cell production process rather thanrelated to the antibody itself.

Example 12 Testing of Humanized Variants of 16C3

Five different designs for converting the murine 16C3 antibody to ahumanized, therapeutically useful antibody are presented in FIG. 5 andFIG. 6. Recombinant humanized 16C3 expressed in 293T cells,co-transfection of five variants in matrix experiment. Supernatants fromthe transient transfection were normalized to 2 ng/mL then diluted toestimate affinity compared to purified recombinant mouse 16C3, thentested for antigen binding potential to AsPC-1 pancreatic tumor cells byFACS, and compared to the recombinant mouse 16C3 antibody. The datashown in Table 7 demonstrates that each of the five variants of heavychain could fold properly with each of the four variant light chains toresult in antigen binding activity. The binding of each combination ofhumanized heavy chain and light chain was comparable to the binding bythe original mouse 16C3 antibody. The data show that humanizing the 16C3immunoglobulin by any of five different methods did not alter theantigen-recognition site of the resulting antibody. The binding of eachwas titratable with the amount of antibody, with each combinationdemonstrating similar titration profiles.

TABLE 7 FACS data from recombinant humanized 16C3. % AsPC-1 tumorAntibody Sample cell binding (MFI) Goat anti-Mouse IgG-FITC control 1.55(70) RECm16C3 cntl., 100 ng 61.66 (228) RECm16C3 cntl., 20 ng 47.91(69)  RECm16C3 cntl., 4 ng 4.26 (52) RECm16C3 cntl., 0.8 ng  1.28 (615)Rabbit anti-Human IgG-FITC control 2.05 (59) (Heavy chain/Light chain)Rh16C3 supe 8: VEN/VEN-100 uL  68.14 (1090) Rh16C3 supe 8: VEN/VEN-20 uL65.93 (240) Rh16C3 supe 8: VEN/VEN-4 uL 46.11 (65)  Rh16C3 supe 10:VEN/ABB-100 uL 64.19 (549) Rh16C3 supe 10: VEN/ABB-20 uL 62.24 (122)Rh16C3 supe 10: VEN/ABB-4 uL 22.10 (52)  Rh16C3 supe 11: VEN/SDR-100 uL67.99 (808) Rh16C3 supe 11: VEN/SDR-20 uL 66.45 (191) Rh16C3 supe 11:VEN/SDR-4 uL 32.30 (57)  Rh16C3 supe 12: VEN/FRA-100 uL 67.22 (674)Rh16C3 supe 12: VEN/FRA-20 uL 64.11 (143) Rh16C3 supe 12: VEN/FRA-4 uL25.65 (51)  Rh16C3 supe 14: CDR/VEN-100 uL 62.66 (568) Rh16C3 supe 14:CDR/VEN-20 uL 59.92 (112) Rh16C3 supe 14: CDR/VEN-4 uL 16.89 (49) Rh16C3 supe 16: CDR/ABB-100 uL 64.49 (254) Rh16C3 supe 16: CDR/ABB-20 uL49.61 (73)  Rh16C3 supe 16: CDR/ABB-4 uL 5.85 (70) Rh16C3 supe 17:CDR/SDR-100 uL 68.02 (376) Rh16C3 supe 17: CDR/SDR-20 uL 54.78 (89) Rh16C3 supe 17: CDR/SDR-4 uL 10.21 (53)  Rh16C3 supe 18: CDR/FRA-100 uL61.54 (557) Rh16C3 supe 18: CDR/FRA-20 uL 57.34 (98)  Rh16C3 supe 18:CDR/FRA-4 uL 17.55 (51)  Rh16C3 supe 20: ABB/VEN-100 uL 65.72 (374)Rh16C3 supe 20: ABB/VEN-20 uL 57.34 (89)  Rh16C3 supe 20: ABB/VEN-4 uL6.39 (54) Rh16C3 supe 22: ABB/ABB-100 uL 66.31 (318) Rh16C3 supe 22:ABB/ABB-20 uL 50.30 (78)  Rh16C3 supe 22: ABB/ABB-4 uL 7.63 (50) Rh16C3supe 23: ABB/SDR-100 uL 66.33 (293) Rh16C3 supe 23: ABB/SDR-20 uL 52.12(75)  Rh16C3 supe 23: ABB/SDR-4 uL 6.58 (59) Rh16C3 supe 24: ABB/FRA-100uL 65.15 (403) Rh16C3 supe 24: ABB/FRA-20 uL 57.63 (98)  Rh16C3 supe 24:ABB/FRA-4 uL 12.29 (50)  Rh16C3 supe 26: SDR/VEN-100 uL 67.94 (495)Rh16C3 supe 26: SDR/VEN-20 uL 62.15 (140) Rh16C3 supe 26: SDR/VEN-4 uL12.69 (59)  Rh16C3 supe 28: SDR/ABB-100 uL 66.58 (314) Rh16C3 supe 28:SDR/ABB-20 uL 54.71 (87)  Rh16C3 supe 28: SDR/ABB-4 uL 8.59 (51) Rh16C3supe 29: SDR/SDR-100 uL 67.95 (503) Rh16C3 supe 29: SDR/SDR-20 uL 61.56(114) Rh16C3 supe 29: SDR/SDR-4 uL 15.87 (56)  Rh16C3 supe 30:SDR/FRA-100 uL 65.87 (702) Rh16C3 supe 30: SDR/FRA-20 uL 64.45 (156)Rh16C3 supe 30: SDR/FRA-4 uL 29.29 (53)  Rh16C3 supe 32: FRA/VEN-100 uL66.03 (585) Rh16C3 supe 32: FRA/VEN-20 uL 63.64 (147) Rh16C3 supe 32:FRA/VEN-4 uL 22.69 (52)  Rh16C3 supe 34: FRA/ABB-100 uL 67.38 (395)Rh16C3 supe 34: FRA/ABB-20 uL 58.89 (99)  Rh16C3 supe 34: FRA/ABB-4 uL12.30 (51)  Rh16C3 supe 35: FRA/SDR-100 uL 68.01 (465) Rh16C3 supe 35:FRA/SDR-20 uL 61.35 (114) Rh16C3 supe 35: FRA/SDR-4 uL 14.23 (59) Rh16C3 supe 36: FRA/FRA-100 uL 67.88 (432) Rh16C3 supe 36: FRA/FRA-20 uL59.15 (99)  Rh16C3 supe 36: FRA/FRA-4 uL 8.68 (53)

Example 13 Optimization and Testing of Recombinant Humanized 16C3Antibody

Additionally, in silico analysis tools are useful in predictingpotential T-cell epitopes in a protein. Such algorithms are useful todesign improved recombinant proteins with a decreased likelihood ofimmunogenicity in humans. To take advantage of this predictivetechnology, the five variant humanized 16C3 heavy chain and light chaingenes were analyzed and shown to harbor one or more predicted T-cellepitopes that might possibly induce an immunogenic response in humanswith certain HLA haplotypes. Such analysis may be done using softwaresuch as Epimer or EpiMatrix, in silico epitope-mapping tools, or by acommercial vendor, such as Antitope, Ltd. (Cambridge, UK). In an attemptto remove these T-cell epitopes to decrease possible immunogenicity of apotential therapeutic 16C3 antibody, specific point mutations were madeto delete the T-cell epitopes and replace specific amino acids thatwould result in a fully functional, but less immunogenic, antibodymolecule. The protein sequence of the optimized humanized 16C3 antibodyis shown in FIG. 12, with the bolded amino acids indicating CDRs, and“/” indicating the leader peptide/mature N-terminus junction and thevariable/constant domain junction.

The genes for the h16C3-Abb* antibody were made by mutagenesis of theexisting variant genes to yield the desired DNA sequences. Then, theh16C3-Abb* heavy and light chain genes were cloned into a mammalianexpression plasmid and transfected into CHO cells. The supernatants ofseveral resulting clones were tested for binding to LS174T (colon) andCFPAC-1 (pancreas) tumor cells by FACS, with Rabbit anti-Human IGG-FITCas the control. The data presented in Table 8 demonstrates that theoptimized, humanized 16C3 (H16C3) gene designs resulted in an antibodywith very good antigen recognition activity. This particular h16C3-Abb*design represents an antibody with high binding activity withpotentially low immunogenicity and/or toxicity as a therapeutic antibodyfor use in cancers that express the target antigen.

TABLE8 Result of FACS experiment on humanized 16C3-Abb* transfectionsupernatants. % binding to LS174T % binding to CFPAC-1 Antibody Samplecells (mfi) cells (mfi) Control 3.49 (33) 2.10 (20) H16C3-Abb* supe 154.40 (374) 98.62 (411) H16C3-Abb* supe 2 51.10 (299) 97.70 (299)H16C3-Abb* supe 3 55.20 (402) 98.60 (486) H16C3-Abb* supe 4 53.75 (333)98.68 (371) H16C3-Abb* supe 5 56.24 (407) 99.14 (447)

The ADCC activity of h16C3-Abb* was tested against pancreatic CFPAC-1and ASPC-1 carcinoma lines as target cells. The melanoma cell line,SK-MEL, served as a tumor cell specificity control. ADCC was assayedusing a conventional four hour ¹¹¹In-release assay using normal humanPBMC as effector cells, and the results are shown as the percent isotoperelease (% lysis) below. Compared to the negative control antibodyUPC-10, the data indicate antibody-specific killing activity by thehumanized 16C3 antibody. The killing activity appeared to be specificfor pancreatic tumor lines since no lysis was observed against themelanoma negative control cells. These data show that the killingactivity of an antibody could be engineered through humanization of themouse 16C3 antibody. Compared to the mouse 16C3 antibody, the humanized16C3 antibody demonstrated superior killing activity, most likely due tothe human Fc region that could interact more efficiently than the mouseFc region with human effector cells.

TABLE 9 ADCC assay with h16C3-Abb* antibody. % Specific % Specific ADCCActivity ADCC Activity Effector:Target (±SEM) (±SEM) Tumor Target Ratioh16C3-Abb* UPC-10 control AsPC-1 100 32.2 ± 0.56 0.4 ± 0.38 (pancreas)50 18.4 ± 2.67 −0.1 ± 0.54   25 14.4 ± 1.66 0.2 ± 0.36 CFPAC-1 100 48.7± 3.22 1.9 ± 0.26 (pancreas) 50 40.9 ± 4.11 2.6 ± 0.49 25 19.4 ± 2.072.1 ± 0.20 SK-MEL 100  0.1 ± 1.28 −0.6 ± 0.18   (melanoma) 50 −1.2 ±0.78 −1.8 ± 0.34   25 0.1 ± 0.2 −1.1 ± 0.83   ¹¹¹In-labeled targetcells, antibodies used at 5 μg/well, IL-2 activated human PBMC used aseffector cells, 4 hour incubation at 37° C. before harvest.

Example 14 Characterization of the CPAA Recognized by 16C3

Several characteristics of the antigen to which the 16C3 antibody bindswere examined in western blots using various treatments. The data inTable 10 demonstrate that the 16C3 antigen is present in some, but notall, cultured colorectal and pancreatic tumor cells. Importantly, the16C3 antigen is present in fetal tissue extracts derived from the gutand intestine. The Fraction I positive specimens represent eluates(Fraction I) from Sephadex G-200 column chromatography runs usingembryonic tissues dissected and subjected to the Hollinshead method ofmembrane protein extraction and purification. The colorectal tumorspecimens were obtained from surgical procedures. The tissues wereminced and subjected to total protein extraction using detergents. Table10 shows data from the expression of 16C3 tumor antigen in various tumorcells by western blot of these cell extracts. These data suggest thatthe 16C3 tumor antigen may be expressed during the embryonic stage oflife as well as in cancer. The expression of the 16C3 antigen,therefore, could be developmentally regulated to be expressed duringembryonic tissue development, and again during cancer development.

TABLE 10 Expression of 16C3 tumor antigen in various tumor cells. 16C3Antigen Cell Line SW1116 (colorectal) Positive (MW ~220 kDa) SW480(colorectal) negative SW1463 (colorectal) negative COLO-205 (colorectal)Positive (MW ~220 kDa) CALU-1 (lung) negative PANC-1 (pancreas) negativePR-22 (prostate) negative HT-29 (colorectal) Positive (MW ~220 kDa and110 kDa) LS174T (colorectal) Positive (MW ~220 kDa and 110 kDa minorCFPAC-1 (pancreas) Positive (MW ~220 kDa and 110 kDa) ASPC-1 (pancreas)Positive (MW ~220 kDa and 110 kDa) Human Tissue Preparation Fetal gut,Fraction I, Positive (MW ~220 kDa and 110 kDa) Dec. 20, 1972 Fetalintestine, Fraction I, Positive (MW ~220 kDa and 110 kDa) Jun. 24, 1975Colorectal tumor tissues, Positive (MW ~220 kDa and 110 kDa) resected

A qualitative description of the 16C3 tumor antigen expressed bycolorectal and pancreatic tumor cell lines was pursued using westernblot analysis following various chemical treatments. These findings arepresented in FIG. 8-FIG. 11, and Table 11.

TABLE 11 Western blot analysis following various chemical treatments.Tumor Cell Line Characteristic LS174T CFPAC ASPC-1 Semi quantitation ofBy western blot By western blot By western blot antigen expression inRelative amount 100% Relative amount 300% Relative amount ~30%supernatant (LS174 is expressed as 100%) Semi quantitation of By westernblot By western blot By western blot antigen expression in Relativeamount 100% Relative amount 200% Relative amount 80% cell pellet (LS174is expressed as 100%) Ratio of presence in cell 100/20 100/20 100/20pellet vs. supernatant ~MW in SDS gel as ~200 kDa and 110 kDa ~200 kDaand 110 kDa ~200 kDa and 110 kDa determined by (minor component) westernblot Effect of reducing agents No effect on antigenicity No effect on Noeffect on DDT or 2-ME or molecular weight antigenicity or antigenicityor molecular weight molecular weight Glycosidase treatment Antigenicityis not effected by glycosidase treatment, but PNGase F reduced themolecular weight of the 200 kDa band to ~130 kDa. The lower (110 kDa)band is not effected by PNGase F Beta elimination Loss of antigenicityusing NaOH Trypsin treatment for 24 Antigenicity not effected butreduction of the Not Done hours at 25° C. or 37° C. molecular weight(diffuse broad band) Protease V8 treatment Antigenicity not effected butreduction of the Not done 24 hours at 25° C. molecular weight (diffusebroad band)

Although this invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications. This application is intended to cover any variations,uses, or adaptations of the inventions following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth as follows in the scope of theappended claims.

1. An isolated recombinant monoclonal antibody comprising: two lightchain components and two heavy chain components wherein each of saidlight chain components contains the amino acids depicted in FIG. 4 (SEQID NO:14), or a mutant, variant, homolog, portion, fragment orderivative of this sequence, and each of said heavy chain componentscontains the amino acids depicted in FIG. 5 (SEQ ID NO: 15), or amutant, variant, homolog, portion, fragment or derivative of thissequence, and wherein said antibody exhibits ADCC activity specific forcolorectal and pancreatic cancer cells.
 2. A diagnostic kit comprisingthe antibody of claim
 1. 3. A composition comprising the antibody ofclaim 1 and a carrier.
 4. The antibody of claim 1, wherein said antibodyis linked to a label.
 5. A chimeric antibody comprising the variableregions of the light and heavy chains of the recombinant antibody asdescribed in claim 1 linked to the human immunoglobulin gamma-1 andkappa constant regions, respectively.
 6. A diagnostic kit comprising theantibody of claim
 5. 7. A composition comprising the antibody of claim 5and a carrier.
 8. The antibody of claim 5, wherein said antibody islinked to a label.
 9. An antibody or portion of an antibody comprising:a light chain complementarity determining region (CDR) 1 consisting ofthe amino acid residues GASENIYGALN (SEQ ID NO:1) or QASENIYGALN (SEQ IDNO:4); a light chain CDR3 consisting of the amino acid residues GASNLAD(SEQ ID NO:2) or GASNLAT (SEQ ID NO:5); a light chain CDR3 consisting ofthe amino acid residues QNVLSSPYT (SEQ ID NO:3) or QQVLSSPYT (SEQ IDNO:6); a heavy chain CDR1 consisting of the amino acid residuesGYTFTDYAMH (SEQ ID NO:7); a heavy chain CDR2 consisting of the aminoacid residues LISTYSGDIKYNQNFKG (SEQ ID NO:8) or ISTYSGDTKYNQNFQG (SEQID NO:10); and a heavy chain CDR3 consisting of the amino acid residuesCDYSGSRYWFAY (SEQ ID NO:9) or GDYSGSRYWFAY (SEQ ID NO:11); or afunctional equivalent of any of the foregoing CDRs such that saidantibody or portion of an antibody binds the antigen recognized by 16C3.10. A diagnostic kit comprising the antibody of claim
 9. 11. Acomposition comprising the antibody of claim 9 and a carrier.
 12. Theantibody of claim 9, wherein said antibody is linked to a label.
 13. Ahumanized monoclonal antibody derived from the sequences shown in FIG. 4(SEQ ID NO:14) and FIG. 5 (SEQ ID NO:15), or FIG. 6 (SEQ ID NO:16-NO:21)and FIG. 7 (SEQ ID NO:22-NO:27), or FIG. 12 (SEQ ID NO:28-NO:29); or amutant, variant, homolog, portion, fragment, or derivative of any ofthese sequences.
 14. A diagnostic kit comprising the antibody of claim13.
 15. A composition comprising the antibody of claim 13 and a carrier.16. The antibody of claim 13, wherein said antibody is linked to alabel.
 17. An isolated oligonucleotide selected from the groupconsisting of at least one of the following: an oligonucleotide with thenucleic acids depicted in FIG. 2 (SEQ ID NO:12), an oligonucleotide witha sequence complementary to the sequence of FIG. 2, an oligonucleotidewith the sequence of FIG. 3 (SEQ ID NO:13), an oligonucleotide with asequence complementary to the sequence of FIG. 3, a mutant, variant,homolog, portion, fragment, and derivative of any of these.
 18. Theoligonucleotide of claim 17, wherein said oligonucleotide is present ina vector.
 19. The isolated oligonucleotide of claim 17, furthercomprising genetic regions to direct expression of said oligonucleotide.20. The oligonucleotide of claim 19, wherein said oligonucleotide ispresent in a vector.
 21. The vector of claim 20, wherein said vector iscontained in a host cell.
 22. The host cell of claim 21, wherein saidhost cell may be used to express the polypeptide(s) encoded by saidoligonucleotides.